Methods of screening for modulators of HIV infection

ABSTRACT

The present invention provides polynucleotides that encode the chemokine receptors 88-2B or 88C and materials and methods for the recombinant production of these two chemokine receptors. Also provided are assays utilizing the polynucleotides which facilitate the identification of ligands and modulators of the chemokine receptors. Receptor fragments, ligands, modulators, and antibodies are useful in the detection and treatment of disease states associated with the chemokine receptors such as atherosclerosis, rheumatoid arthritis, tumor growth suppression, asthma, viral infection, AIDS, and other inflammatory conditions.

[0001] This is a continuation application of U.S. patent applicationSer. No. 08/771,276 filed Dec. 20, 1996, which is a continuation-in-partof U.S. patent application Ser. No. 08/661,393 filed Jun. 7, 1996(issued as U.S. Pat. No. 6,268,477 on Jul. 31, 2001), which was in turna continuation-in-part of U.S. patent application No. 08/575,967 filedDec. 20, 1995 (issued as U.S. Pat. No. 6,265,184 on Jul. 24, 2001). Allof these priority applications are incorporated by reference in theirentirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to signal transductionpathways. More particularly, the present invention relates to chemokinereceptors, nucleic acids encoding chemokine receptors, chemokinereceptor ligands, modulators of chemokine receptor activity, antibodiesrecognizing chemokines and chemokine receptors, methods for identifyingchemokine receptor ligands and modulators, methods for producingchemokine receptors, and methods for producing antibodies recognizingchemokine receptors.

BACKGROUND OF THE INVENTION

[0003] Recent advances in molecular biology have led to an appreciationof the central role of signal transduction pathways in biologicalprocesses. These pathways comprise a central means by which individualcells in a multicellular organism communicate, thereby coordinatingbiological processes. See Springer, Cell 76:301-314 (1994), Table I, fora model. One branch of signal transduction pathways, defined by theintracellular participation of guanine nucleotide binding proteins(G-proteins), affects a broad range of biological processes.

[0004] Lewin, GENES V 319-348 (1994) generally discusses G-proteinsignal transduction pathways which involve, at a minimum, the followingcomponents: an extracellular signal (e.g., neurotransmitters, peptidehormones, organic molecules, light, or odorants), a signal-recognizingreceptor [G-protein-coupled receptor, reviewed in Probst et al., DNA andCell Biology 11:1-20 (1992) and also known as GPR or GPCR], and anintracellular, heterotrimeric GTP-binding protein, or G protein. Inparticular, these pathways have attracted interest because of their rolein regulating white blood cell or leukocyte trafficking.

[0005] Leukocytes comprise a group of mobile blood cell types includinggranulocytes (i.e., neutrophils, basophils, and eosinophils),lymphocytes, and monocytes. When mobilized and activated, these cellsare primarily involved in the body's defense against foreign matter.This task is complicated by the diversity of normal and pathologicalprocesses in which leukocytes participate. For example, leukocytesfunction in the normal inflammatory response to infection. Leukocytesare also involved in a variety of pathological inflammations. For asummary, see Schall et al., Curr. Opin. Immunol. 6:865-873 (1994).Moreover, each of these processes can involve unique contributions, indegree, kind, and duration, from each of the leukocyte cell types.

[0006] In studying these immune reactions, researchers initiallyconcentrated on the signals acting upon leukocytes, reasoning that asignal would be required to elicit any form of response. Murphy, Ann.Rev. Immunol. 12:593-633 (1994) has reviewed members of an importantgroup of leukocyte signals, the peptide signals. One type of peptidesignal comprises the chemokines (chemoattractant cytokines), termedintercrines in Oppenheim et al., Ann. Rev. Immunol. 9:617-648 (1991). Inaddition to Oppenheim et al., Baggiolini et al., Advances in Immunol.55:97-179 (1994), documents the growing number of chemokines that havebeen identified and subjected to genetic and biochemical analyses.

[0007] Comparisons of the amino acid sequences of the known chemokineshave led to a classification scheme which divides chemokines into twogroups: the a group characterized by a single amino acid separating thefirst two cysteines (CXC; N-terminus as referent), and the βgroup, wherethese cysteines are adjacent (CC). See Baggiolini et al., supra.Correlations have been found between the chemokines and the particularleukocyte cell types responding to those signals. Schall et al., supra,has reported that the CXC chemokines generally affect neutrophils; theCC chemokines tend to affect monocytes, lymphocytes, basophils andeosinophils. For example, Baggiolini et al., supra, recited that RANTES,a CC chemokine, functions as a chemoattractant for monocytes,lymphocytes (i.e., memory T cells), basophils, and eosinophils, but notfor neutrophils, while inducing the release of histamine from basophils.

[0008] Chemokines were recently shown by Cocchi et. al., Science,270:1811-1815 (1995) to be suppressors of HIV proliferation. Cocchi etal. (supra) demonstrated that RANTES, MIP-1α, and MIP-1β suppressedHIV-1, HIV-2 and SIV infection of a CD4₊cell line designated PM1 and ofprimary human peripheral blood mononuclear cells.

[0009] Recently, however, attention has turned to the cellular receptorsthat bind the chemokines, because the extracellular chemokines seem tocontact cells indiscriminately, and therefore lack the specificityneeded to regulate the individual leukocyte cell types.

[0010] Murphy (supra) reported that the GPCR superfamily of receptorsincludes the chemokine receptor family. The typical chemokine receptorstructure includes an extracellular chemokine-binding domain locatednear the N-terminus, followed by seven spaced regions of predominantlyhydrophobic amino acids capable of forming membrane-spanning α-helices.Between each of the a-helical domains are hydrophilic domains localized,alternately, in the intra- or extra-cellular spaces. These featuresimpart a serpentine conformation to the membrane-embedded chemokinereceptor. The third intracellular loop typically interacts withG-proteins. In addition, Murphy (supra) noted that the intracellularcarboxyl terminus is also capable of interacting with G-proteins.

[0011] The first chemokine receptors to be analyzed by molecular cloningtechniques were the two neutrophil receptors for human IL8, a CXCchemokine. Holmes et al., Science 253:178-1280 (1991) and Murphy et al.,Science 253:1280-1283 (1991), reported the cloning of these tworeceptors for IL8. Lee et al., J. Biol. Chem. 267:16283-16287 (1992),analyzed the cDNAs encoding these receptors and found 77% amino acididentity between the encoded receptors, with each receptor exhibitingfeatures of the G protein coupled receptor family. One of thesereceptors is specific for IL-8, while the other binds and signals inresponse to IL-8, gro/MGSA, and NAP-2. Genetic manipulation of the genesencoding IL-8 receptors has contributed to our understanding of thebiological roles occupied by these receptors. For example, Cacalano etal., Science 265:682-684 (1994) reported that deletion of the IL-8receptor homolog in the mouse resulted in a pleiotropic phenotypeinvolving lymphadenopathy and splenomegaly. In addition, a study ofmissense mutations described in Leong et al., J. Biol. Chem.269:19343-19348 (1994) revealed amino acids in the IL-8 receptor thatwere critical for IL-8 binding. Domain swapping experiments discussed inMurphy (supra) implicated the amino terminal extracellular domain as adeterminant of binding specificity.

[0012] Several receptors for CC chemokines have also been identified andcloned. CCCKR1 binds both MIP-1α and RANTES and causes intracellularcalcium ion flux in response to both ligands. Charo et al., Proc Natl.Acad. Sci. (USA) 91:2752-2756 (1994) reported that another CC chemokinereceptor, MCP-R1 (CCCKR2), is encoded by a single gene that produces twosplice variants which differ in their carboxyl terminal domains. Thisreceptor binds and responds to MCP-3 in addition to MCP-1.

[0013] A promiscuous receptor that binds both CXC and CC chemokines hasalso been identified. This receptor was originally identified on redblood cells and Horuk et al., Science 261:1182-1184 (1993) reports thatit binds IL-8, NAP-2, GROα, RANTES, and MCP-1. The erythrocyte chemokinereceptor shares about 25% identity with other chemokine receptors andmay help to regulate circulating levels of chemokines or aid in thepresentation of chemokines to their targets. In addition to bindingchemokines, the erythrocyte chemokine receptor has also been shown to bethe receptor for plasmodium vivax, a major cause of malaria (id.)Another G-protein coupled receptor which is closely related to chemokinereceptors, the platelet activating factor receptor, has also been shownto be the receptor for a human pathogen, the bacterium Streptococcuspneumoniae [Cundell et al., Nature 377:435-438 (1995)].

[0014] In addition to the mammalian chemokine receptors, two viralchemokine receptor homologs have been identified. Ahuja et al., J. Biol.Chem. 268:20691-20694 (1993) describes a gene product from Herpesvirussaimiri that shares about 30% identity with the IL-8 receptors and bindsCXC chemokines. Neote et al., Cell, 72:415-425 (1993) reports that humancytomegalovirus contains a gene encoding a receptor sharing about 30%identity with the CC chemokine receptors which binds MIP-1α, MIP-1β,MCP-1, and RANTES. These viral receptors may affect the normal role ofchemokines and provide a selective pathological advantage for the virus.

[0015] Because of the broad diversity of chemokines and theiractivities, there are numerous receptors for the chemokines. Thereceptors which have been characterized represent only a fraction of thetotal complement of chemokine receptors. There thus remains a need inthe art for the identification of additional chemokine receptors. Theavailability of these novel receptors will provide tools for thedevelopment of therapeutic modulators of chemokine or chemokine receptorfunction. It is contemplated by the present invention that suchmodulators are useful as therapeutics for the treatment ofatherosclerosis, rheumatoid arthritis, tumor growth suppression, asthma,viral infections, and other inflammatory conditions. Alternatively,fragments or variants of the chemokine receptors, or antibodiesrecognizing those receptors, are contemplated as therapeutics.

SUMMARY OF THE INVENTION

[0016] The present invention provides purified and isolated nucleicacids encoding chemokine receptors involved in leukocyte trafficking.Polynucleotides of the invention (both sense and anti-sense strandsthereof) include genomic DNAs, cDNAs, and RNAs, as well as completely orpartially synthetic nucleic acids. Preferred polynucleotides of theinvention include the DNA encoding the chemokine receptor 88-2B that isset out in SEQ ID NO:3, the DNA encoding the chemokine receptor 88C thatis set out in SEQ ID NO: 1, and DNAs which hybridize to those DNAs understandard stringent hybridization conditions, or which would hybridizebut for the redundancy of the genetic code. Exemplary stringenthybridization conditions are as follows: hybridization at 42° C. in 50%formamide, 5X SSC, 20 mM sodium phosphate, pH 6.8 and washing in 0.2XSSC at 55° C.

[0017] It is understood by those of skill in the art that variation inthese conditions occurs based on the length and GC nucleotide content ofthe sequences to be hybridized. Formulas standard in the art areappropriate for determining exact hybridization conditions. See Sambrooket al., §§ 9.47-9.51 in Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y (1989). Alsocontemplated by the invention are polynucleotides encoding domains of88-2B or 88C, for example, polynucleotides encoding one or moreextracellular domains of either protein or other biologically activefragments thereof. 88-2B extracellular domains correspond to SEQ ID NO:3and SEQ ID NO:4 at amino acid residues 1-36, 93-107, 171-196, and263-284. The extracellular domains of 88-2B are encoded bypolynucleotide sequences corresponding to SEQ ID NO:3 at nucleotides362-469, 638-682, 872-949, and 1148-1213. Extracellular domains of 88Ccorrespond to SEQ ID NO: 1 and SEQ ID NO:2 at amino acid residues 1-32,89-112, 166-191, and 259-280. The 88C extracellular domains are encodedby polynucleotide sequences that correspond to SEQ ID NO: 1 atnucleotides 55-150, 319-390, 550-627, and 829-894. The invention alsocomprehends polynucleotides encoding intracellular domains of thesechemokine receptors. The intracellular domains of 88-2B include aminoacids 60-71, 131-151, 219-240, and 306-355 of SEQ ID NO:3 and SEQ IDNO:4. Those domains are encoded by polynucleotide sequencescorresponding to SEQ ID NO:3 at nucleotides 539-574, 752-814, 1016-1081,and 1277-1426, respectively. The 88C intracellular domains include aminoacid residues 56-67, 125-145, 213-235, and 301-352 of SEQ ID NO: 1 andSEQ ID NO:2. The intracellular domains of 88C are encoded bypolynucleotide sequences corresponding to SEQ ID NO: 1 at nucleotides220-255, 427-489, 691-759, and 955-1110. Peptides corresponding to oneor more of the extracellular or intracellular domains, or antibodiesraised against those peptides, are contemplated as modulators ofreceptor activities, especially ligand and G protein binding activitiesof the receptors.

[0018] The nucleotide sequences of the invention may also be used todesign oligonucleotides for use as labeled probes to isolate genomicDNAs encoding 88-2B or 88C under stringent hybridization conditions(i.e., by Southern analyses and polymerase chain reactionmethodologies). Moreover, these oligonucleotide probes can be used todetect particular alleles of the genes encoding 88-2B or 88C,facilitating both diagnosis and gene therapy treatments of diseasestates associated with particular alleles. In addition, theseoligonucleotides can be used to alter chemokine receptor genetics tofacilitate identification of chemokine receptor modulators. Also, thenucleotide sequences can be used to design antisense genetic elements ofuse in exploring or altering the genetics and expression of 88-2B or88C. The invention also comprehends biological replicas (i.e., copies ofisolated DNAs made in vivo or in vitro) and RNA transcripts of DNAs ofthe invention.

[0019] Autonomously replicating recombinant constructions such asplasmid, viral, and chromosomal (e.g., YAC) nucleic acid vectorseffectively incorporating 88-2B or 88C polynucleotides, and,particularly, vectors wherein DNA effectively encoding 88-2B or 88C isoperatively linked to one or more endogenous or heterologous expressioncontrol sequences are also provided.

[0020] The 88-2B and 88C receptors may be produced naturally,recombinantly or synthetically. Host cells (prokaryotic or eukaryotic)transformed or transfected with polynucleotides of the invention bystandard methods may be used to express the 88-2B and 88C chemokinereceptors. Beyond the intact 88-2B or 88C gene products, biologicallyactive fragments of 88-2B or 88C, analogs of 88-2B or 88C, and syntheticpeptides derived from the amino acid sequences of 88-2B, set out in SEQID NO:4, or 88C, set out in SEQ ID NO:2, are contemplated by theinvention. Moreover, the 88-2B or 88C gene product, or a biologicallyactive fragment of either gene product, when produced in a eukaryoticcell, may be post-translationally modified (e.g., via disulfide bondformation, glycosylation, phosphorylation, myristoylation,palmitoylation, acetylation, etc.) The invention further contemplatesthe 88-2B and 88C gene products, or biologically active fragmentsthereof, in monomeric, homomultimeric, or heteromultimericconformations.

[0021] In particular, one aspect of the invention involves antibodyproducts capable of specifically binding to the 88-2B or 88C chemokinereceptors. The antibody products are generated by methods standard inthe art using recombinant 88-2B or 88C receptors, synthetic peptides orpeptide fragments of 88-2B or 88C receptors, host cells expressing 88-2Bor 88C on their surfaces, or 88-2B or 88C receptors purified fromnatural sources as immunogens. The antibody products may includemonoclonal antibodies or polyclonal antibodies of any source orsub-type. Moreover, monomeric, homomultimeric, and heteromultimericantibodies, and fragments thereof, are contemplated by the invention.Further, the invention comprehends CDR-grafted antibodies, “humanized”antibodies, and other modified antibody products retaining the abilityto specifically bind a chemokine receptor.

[0022] The invention also contemplates the use of antibody products fordetection of the 88-2B or 88C gene products, their analogs, orbiologically active fragments thereof. For example, antibody productsmay be used in diagnostic procedures designed to reveal correlationsbetween the expression of 88-2B, or 88C, and various normal orpathological states. In addition, antibody products can be used todiagnose tissue-specific variations in expression of 88-2B or 88C, theiranalogs, or biologically active fragments thereof.

[0023] Antibody products specific for the 88-2B and 88C chemokinereceptors may also act as modulators of receptor activities. In anotheraspect, antibodies to 88-2B or 88C receptors are useful for therapeuticpurposes.

[0024] Assays for ligands capable of interacting with the chemokinereceptors of the invention are also provided. These assays may involvedirect detection of chemokine receptor activity, for example, bymonitoring the binding of a labeled ligand to the receptor. In addition,these assays may be used to indirectly assess ligand interaction withthe chemokine receptor. As used herein the term “ligand” comprisesmolecules which are agonists and antagonists of 88-2B or 88C, and othermolecules which bind to the receptors.

[0025] Direct detection of ligand binding to a chemokine receptor may beachieved using the following assay. Test compounds (i.e., putativeligands) are detectably labeled (e.g., radioiodinated). The detectablylabeled test compounds are then contacted with membrane preparationscontaining a chemokine receptor of the invention. Preferably, themembranes are prepared from host cells expressing chemokine receptors ofthe invention from recombinant vectors. Following an incubation periodto facilitate contact between the membrane-embedded chemokine receptorsand the detectably labeled test compounds, the membrane material iscollected on filters using vacuum filtration. The detectable labelassociated with the filters is then quantitated. For example,radiolabels are quantitated using liquid scintillationspectrophotometry. Using this technique, ligands binding to chemokinereceptors are identified. To confirm the identification of a ligand, adetectably labeled test compound is exposed to a membrane preparationdisplaying a chemokine receptor in the presence of increasing quantitiesof the test compound in an unlabeled state. A progressive reduction inthe level of filter-associated label as one adds increasing quantitiesof unlabeled test compound confirms the identification of that ligand.

[0026] Agonists are ligands which bind to the receptor and elicitintracellular signal transduction and antagonists are ligands which bindto the receptor but do not elicit intracellular signal transduction. Thedetermination of whether a particular ligand is an agonist or anantagonist can be determined, for example, by assaying G protein-coupledsignal transduction pathways. Activation of these pathways can bedetermined by measuring intracellular ca++ flux, phospholipase Cactivity or adenylyl cyclase activity, in addition to other assays (seeexamples 5 and 6).

[0027] As discussed in detail in the Examples herein, chemokines thatbind to the 88C receptor include RANTES, MIP-1α, and MIP-1β, andchemokines that bind to the 88-2B receptor include RANTES.

[0028] In another aspect, modulators of the interaction between the 88Cand 88-2B receptors and their ligands are specifically contemplated bythe invention. Modulators of chemokine receptor function may beidentified using assays similar to those used for identifying ligands.The membrane preparation displaying a chemokine receptor is exposed to aconstant and known quantity of a detectably labeled functional ligand.In addition, the membrane-bound chemokine receptor is also exposed to anincreasing quantity of a test compound suspected of modulating theactivity of that chemokine receptor. If the levels of filter-associatedlabel correlate with the quantity of test compound, that compound is amodulator of the activity of the chemokine receptor. If the level offilter-associated label increases with increasing quantities of the testcompound, an activator has been identified.

[0029] In contrast, if the level of filter-associated label variesinversely with the quantity of test compound, an inhibitor of chemokinereceptor activity has been identified. Testing for modulators ofreceptor binding in this way allows for the rapid screening of manyputative modulators, as pools containing many potential modulators canbe tested simultaneously in the same reaction.

[0030] The indirect assays for receptor binding involve measurements ofthe concentration or level of activity of any of the components found inthe relevant signal transduction pathway. Chemokine receptor activationoften is associated with an intracellular Ca++ flux. Cells expressingchemokine receptors may be loaded with a calcium-sensitive dye. Uponactivation of the expressed receptor, a Ca++ flux would be renderedspectrophotometrically detectable by the dye. Alternatively, the Ca++flux could be detected microscopically. Parallel assays, using eithertechnique, may be performed in the presence and absence of putativeligands. For example, using the microscopic assay for Ca++ flux, RANTES,a CC chemokine, was identified as a ligand of the 88-2B chemokinereceptor. Those skilled in the art will recognize that these assays arealso useful for identifying and monitoring the purification ofmodulators of receptor activity. Receptor activators and inhibitors willactivate or inhibit, respectively, the interaction of the receptors withtheir ligands in these assays.

[0031] Alternatively, the association of chemokine receptors with Gproteins affords the opportunity of assessing receptor activity bymonitoring G protein activities. A characteristic activity of Gproteins, GTP hydrolysis, may be monitored using, for example,³²P-labeled GTP.

[0032] G proteins also affect a variety of other molecules through theirparticipation in signal transduction pathways. For example, G proteineffector molecules include adenylyl cyclase, phospholipase C, ionchannels, and phosphodiesterases. Assays focused on any of theseeffectors may be used to monitor chemokine receptor activity induced byligand binding in a host cell that is both expressing the chemokinereceptor of interest and contacted with an appropriate ligand. Forexample, one method by which the activity of chemokine receptors may bedetected involves measuring phospholipase C activity. In this assay, theproduction of radiolabeled inositol phosphates by host cells expressinga chemokine receptor in the presence of an agonist is detected. Thedetection of phospholipase activity may require cotransfection with DNAencoding an exogenous G protein. When cotransfection is required, thisassay can be performed by cotransfection of chimeric G protein DNA, forexample, Gqi5 [Conklin et al., Nature 363:274-276 (1993)], with 88-2B or88C DNA and detecting phosphoinositol production when the cotransfectedcell is exposed to an agonist of the 88-2B or 88C receptor. Thoseskilled in the art will recognize that assays focused on G-proteineffector molecules are also useful for identifying and monitoring thepurification of modulators of receptor activity. Receptor activators andinhibitors will activate or inhibit, respectively, the interaction ofthe receptors with their ligands in these assays.

[0033] Chemokines have been linked to many inflammatory diseases, suchas psoriasis, arthritis, pulmonary fibrosis and atherosclerosis. SeeBaggiolini et al. (supra).

[0034] Inhibitors of chemokine action may be useful in treating theseconditions. In one example, Broaddus et al., J. of Immunol.152:2960-2967 (1994), describes an antibody to IL-8 which can inhibitneutrophil recruitment in endotoxin-induced pleurisy, a model of acuteinflammation in rabbit lung. It is also contemplated that ligand ormodulator binding to, or the activation of, the 88C receptor may beuseful in treatment of HIV infection and HIV related disease states.Modulators of chemokine binding to specific receptors contemplated bythe invention may include antibodies directed toward a chemokine or areceptor, biological or chemical small molecules, or synthetic peptidescorresponding to fragments of the chemokine or receptor.

[0035] Administration of compositions containing 88-2B or 88C modulatorsto mammalian subjects, for the purpose of monitoring or remediatingnormal or pathological immune reactions And viral infections includinginfection by retroviruses such as HIV-1, HIV-2 and SIV is contemplatedby the invention. In particular, the invention comprehends themitigation of inflammatory responses, abnormal hematopoietic processes,and viral infections by delivery of a pharmaceutically acceptablequantity of 88-2B or 88C chemokine receptor modulators. The inventionfurther comprehends delivery of these active substances inpharmaceutically acceptable compositions comprising carriers, diluents,or medicaments. The invention also contemplates a variety ofadministration routes. For example, the active substances may beadministered by the following routes: intravenous, subcutaneous,intraperitoneal, intramuscular, oral, anal (i.e., via suppositoryformulations), or pulmonary (i.e., via inhalers, atomizers, nebulizers,etc.)

[0036] In another aspect, the DNA sequence information provided by thepresent invention makes possible the development, by homologousrecombination or “knockout” strategies [see, e.g. Kapecchi, Science,244:1288-1292 (1989)], of rodents that fail to express a functional 88Cor 88-2B chemokine receptor or that express a variant of the receptor.Alternatively, transgenic mice which express a cloned 88-2B or 88Creceptor can be prepared by well known laboratory techniques[Manipulating the Mouse Embryo: A Laboratory Manual, Brigid Hohan, FrankCostantini and Elizabeth Lacy, eds. (1986) Cold Spring Harbor LaboratoryISBN 0-87969-175-I]. Such rodents are useful as models for studying theactivities of 88C or 88-2B receptors in vivo.

[0037] Other aspects and advantages of the present invention will becomeapparent to one skilled in the art upon consideration of the followingexamples.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The following examples illustrate the invention. Example 1describes the isolation of genomic DNAs encoding the 88-2B and 88Cchemokine receptors. Example 2 presents the isolation and sequencing ofcDNAs encoding human 88-2B and 88C and macaque 88C. Example 3 provides adescription of Northern analyses revealing the expression patterns ofthe 88-2B and 88C receptors in a variety of tissues. Example 4 detailsthe recombinant expression of the 88-2B and 88C receptors. Example 5describes Ca++ flux assays, phosphoinositol hydrolysis assays, andbinding assays for 88-2B and 88C receptor activity in response to avariety of potential ligands. Experiments describing the role of 88C and882B as co-receptors for HIV is presented in Examples 6 and 7. Thepreparation and characterization of monoclonal and polyclonal antibodiesimmunoreactive with 88C is described in Example 8. Example 9 describesadditional assays designed to identify 88-2B or 88C ligands ormodulators.

Example 1

[0039] Partial genomic clones encoding the novel chemokine receptorgenes of this invention were isolated by PCR based on conservedsequences found in previously identified genes and based on a clusteringof these chemokine receptor genes within the human genome. The genomicDNA was amplified by standard PCR methods using degenerateoligonucleotide primers.

[0040] Templates for PCR amplifications were members of a commerciallyavailable source of recombinant human genomic DNA cloned into YeastArtificial Chromosomes (i.e., YACs) (Research Genetics, Inc.,Huntsville, Ala., YAC Library Pools, catalog no. 95011 B). A YAC vectorcan accommodate inserts of 500-1000 kilobase pairs. Initially, pools ofYAC clone DNAs were screened by PCR using primers specific for the geneencoding CCCKR1. In particular, CCCKR(2)-5′, the sense strand primer(corresponding to the sense strand of CCCKR1), is presented in SEQ IDNO: 15. Primer CCCKR(2)-5′ consisted of the sequence5′-CGTAAGCTTAGAGAAGCCGGGATGGGAA-3′, wherein the underlined nucleotidesare the translation start codon for CCCKR1. The anti-sense strand primerwas CCCKR-3′ (corresponding to the anti-sense strand of CCCKR1) and itssequence is presented in SEQ ID NO:16. The sequence of CCCKR-3′,5′-GCCTCTAGAGTCAGAGACCAGCAGA-3′, contains the reverse complement of theCCCKR1 translation stop codon (underlined).

[0041] Pools of YAC clone DNAs yielding detectable PCR products (i.e.,DNA bands upon gel electrophoresis) identified appropriate sub-pools ofYAC clones, based on a proprietary identification scheme (ResearchGenetics, Inc., Huntsville, Ala.). PCR reactions were initiated with anincubation at 94° C. for four minutes. Sequence amplifications wereachieved using 33 cycles of denaturation at 94° C. for one minute,annealing at 55° C. for one minute, and extension at 72° C. for twominutes.

[0042] The sub-pools of YAC clone DNAs were then subjected to a secondround of PCR reactions using the conditions, and primers, that were usedin the first round of PCR. Results from sub-pool screenings identifiedindividual clones capable of supporting PCR reactions with theCCCKR-specific primers. One clone, 881F10, contained 640 kb of humangenomic DNA from chromosome 3p21 including the genes for CCCKR1 andCCCKR2, as determined by PCR and hybridization. An overlapping YACclone, 941A7, contained 700 kb of human genomic DNA and also containedthe genes for CCCKR1 and CCCKR2. Consequently, further mapping studieswere undertaken using these two YAC clones. Southern analyses revealedthat CCCKR1 and CCCKR2 were located within approximately 100 kb of oneanother.

[0043] The close proximity of the CCCKR1 and CCCKR2 genes suggested thatnovel related genes might be linked to CCCKR1 and CCCKR2. Using DNA fromyeast containing YAC clones 881F10 and 941A7 as templates, PCR reactionswere performed to amplify any linked receptor genes. Degenerateoligodeoxyribonucleotides were designed as PCR primers. Theseoligonucleotides corresponded to regions encoding the secondintracellular loop and the sixth transmembrane domain of CC chemokinereceptors, as deduced from aligned sequence comparisons of CCCKR1,CCCKR2, and V28. V28 was used because it is an orphan receptor thatexhibits the characteristics of a chemokine receptor; V28 has also beenmapped to human chromosome 3 [Raport et al., Gene 163:295-299 (1995)].Of further note, the two splice variants of CCCKR2, CCCKR2A and CCCKR2B,are identical in the second intracellular loop and sixth transmembranedomain regions used in the analysis. The 5′ primer, designated V28degf2,contains an internal BamHI site (see below); its sequence is presentedin SEQ ID NO:5. The sequence of primer V28degf2 corresponds to DNAencoding the second intracellular loop region of the canonical receptorstructure. See Probst et al., supra. The 3′ primer, designated V28degr2,contains an internal HindIII site (see below); its sequence is presentedin SEQ ID NO:6. The sequence of primer V28degr2 corresponds to DNAencoding the sixth transmembrane domain of the canonical receptorstructure.

[0044] Amplified PCR DNA was subsequently digested with BamHI andHindIII to generate fragments of approximately 390 bp, consistent withthe fragment size predicted from inspection of the canonical sequence.Following endonuclease digestion, these PCR fragments were cloned intopBluescript (Stratagene Inc., LaJolla, Calif.). A total of 54 clonedfragments were subjected to automated nucleotide sequence analyses. Inaddition to sequences from CCCKR1 and CCCKR2, sequences from the twonovel chemokine receptor genes of the invention were identified. Thesetwo novel chemokine receptor genes were designated 88-2B and 88C.

[0045] Restriction endonuclease mapping and hybridization were utilizedto map the relative positions of genes encoding the receptors 88C,88-2B, CCCKR1, and CCCKR2. These four genes are closely linked, as thegene for 88C is approximately 18 KBP from the CCCKR2 gene on humanchromosome 3p21.

EXAMPLE 2

[0046] Full-length 88-2B and 88C cDNAs were isolated from a macrophagecDNA library by the following procedure. Initially, a cDNA library,described in Tjoelker et al., Nature 374:549-553 (1995), was constructedin pRc/CMV (Invitrogen Corp., San Diego, Calif.) from human macrophageMRNA. The cDNA library was screened for the presence of 88-2B and 88CcDNA clones by PCR using unique primer pairs corresponding to 88-2B or88C. The PCR protocol involved an initial denaturation at 94° C. forfour minutes. Polynucleotides were then amplified using 33 cycles of PCRunder the following conditions: Denaturation at 94° C. for one minute,annealing at 55° C. for one minute, and extension at 72° C. for twominutes. The first primer specific for 88-2B was primer 88-2B-f1,presented in SEQ ID NO:11. It corresponds to the sense strand of SEQ IDNO:3 at nucleotides 844-863. The second PCR primer specific for the geneencoding 88-2B was primer 88-2B-r1, presented in SEQ ID NO: 12; the88-2B-r1 sequence corresponds to the anti-sense strand of SEQ ID NO:3 atnucleotides 1023-1042. Similarly, the sequence of the first primerspecific for the gene encoding 88C, primer 88C-f1, is presented in SEQID NO: 13 and corresponds to the sense strand of SEQ ID NO: 1 atnucleotides 453-471. The second primer specific for the gene encoding88C is primer 88C-r3, presented in SEQ ID NO: 14; the sequence of 88C-r3corresponds to the anti-sense strand of SEQ ID NO: 1 at nucleotides744-763.

[0047] The screening identified clone 777, a cDNA clone of 88-2B. Clone777 contained a DNA insert of 1915 bp including the full length codingsequence of 88-2B as determined by the following criteria: the clonecontained a long open reading frame beginning with an ATG codon,exhibited a Kozak sequence, and had an in-frame stop codon upstream. TheDNA and deduced amino acid sequences of the insert of clone 777 arepresented in SEQ ID NO:3 and SEQ ID NO:4, respectively. The 88-2Btranscript was relatively rare in the macrophage cDNA library. Duringthe library screen, only three 88-2B clones were identified from anestimated total of three million clones.

[0048] Screening for cDNA clones encoding the 88C chemokine receptoridentified clones 101 and 134 which appeared to contain the entire 88Ccoding region, including a putative initiation codon. However, theseclones lacked the additional 5′ sequence needed to confirm the identityof the initiation codon. The 88C transcript was relatively abundant inthe macrophage cDNA Library. During the library screen, it was estimatedthat 88C was present at one per 3000 transcripts (in a total ofapproximately three million clones in the library).

[0049] RACE PCR (Rapid Amplification of cDNA Ends) was performed toextend existing 88C clone sequences, thereby facilitating the accuratecharacterization of the 5′ end of the 88C cDNA. Human spleen5′-RACE-ready cDNA was purchased from Clontech Laboratories, Inc., PaloAlto, Calif., and used according to the manufacturer's recommendations.The cDNA had been made “5′ -RACE-ready” by ligating an anchor sequenceto the 5′ ends of the cDNA fragments. The anchor sequence iscomplementary to an anchor primer supplied by Clontech Laboratories,Inc., Palo Alto, Calif. The anchor sequence-anchor primer duplexpolynucleotide contains an EcoRI site. Human spleen cDNA was chosen astemplate DNA because Northern blots had revealed that 88C was expressedin this tissue. The PCR reactions were initiated by denaturing samplesat 94° C. for four minutes. Subsequently, sequences were amplified using35 cycles involving denaturation at 94° C. for one minute, annealing at60° C. for 45 seconds, and extension at 72° C. for two minutes. Thefirst round of PCR was performed on reaction mixtures containing 2 μl ofthe 5′-RACE-ready spleen cDNA, 1 μl of the anchor primer, and 1 μl ofprimer 88c-r4 (100 ng/μl) in a total reaction volume of 50 μl. The88C-specific primer, primer 88c-r4 (5′-GATAAGCCTCACAGCCCTGTG-3′), ispresented in SEQ ID NO:7. The sequence of primer 88c-r4 corresponds tothe anti-sense strand of SEQ ID NO: 1 at nucleotides 745-765. A secondround of PCR was performed on reaction mixtures including 1 μl of thefirst PCR reaction with 1 μl of anchor primer and 1 μl of primer 88C-rlb(100 ng/μl) containing the following sequence(5′-GCTAAGCTTGATGACTATCTTTAATGTC-3′) and presented in SEQ ID NO:8. Thesequence of primer 88C-rlb contains an internal HindIII cloning site(underlined). The sequence 3′ of the HindIII site corresponds to theanti-sense strand of SEQ ID NO:1 at nucleotides 636-654. The resultingPCR product was digested with EcoRI and HindIII and fractionated on a 1%agarose gel. The approximately 700 bp fragment was isolated and clonedinto pBluescript. Clones with the largest inserts were sequenced.Alternatively, the intact PCR product was ligated into vector pCR usinga commercial TA cloning kit (Invitrogen Corp., San Diego, Calif.) forsubsequent nucleotide sequence determinations.

[0050] The 88-2B and 88C cDNAs were sequenced using the PRISM™ ReadyReaction DyeDeoxy™ Terminator Cycle Sequencing Kit (Perkin Elmer Corp.,Foster City, Calif.) and an Applied Biosystems 373A DNA Sequencer. Theinsert of clone 777 provided the double-stranded template for sequencingreactions used to determine the 88-2B cDNA sequence. The sequence of theentire insert of clone 777 was determined and is presented as the 88-2BcDNA sequence and deduced amino acid sequence in SEQ ID NO:3. Thesequence is 1915 bp in length, including 361 bp of 5′ untranslated DNA(corresponding to SEQ ID NO:3 at nucleotides 1-361), a coding region of1065 bp (corresponding to SEQ ID NO:3 at nucleotides 362-1426), and 489bp of 3′ untranslated DNA (corresponding to SEQ ID NO:3 at nucleotides1427-1915). The 88-2B genomic DNA, described in Example 1 above,corresponds to SEQ ID NO:3 at nucleotides 746-1128. The 88C cDNAsequence, and deduced amino acid sequence, is presented in SEQ ID NO: 1.The 88C cDNA sequence is a composite of sequences obtained from RACE-PCRcDNA, clone 134, and clone 101. The RACE-PCR cDNA was used as asequencing template to determine nucleotides 1-654 in SEQ ID NO: 1,including the unique identification of 9 bp of 5′ untranslated cDNAsequence in SEQ ID NO: 1 at nucleotides 1-9. The sequence obtained fromthe RACE PCR cDNA confirmed the position of the first methionine codonat nucleotides 55-57 in SEQ ID NO: 1, and supported the conclusion thatclone 134 and clone 101 contained full-length copies of the 88C codingregion. Clone 134 contained 45 bp of 5′ untranslated cDNA (correspondingto SEQ ID NO: 1 at nucleotides 10-54), the 1056 bp 88C coding region(corresponding to SEQ ID NO:1 at nucleotides 55-1110), and 492 bp of 3′untranslated cDNA (corresponding to SEQ ID NO:1 at nucleotides1111-1602). Clone 101 contained 25 bp of 5′ untranslated cDNA(corresponding to SEQ ID NO: 1 at nucleotides 30-54), the 1056 bp 88Ccoding region (corresponding to SEQ ID NO: 1 at nucleotides 55-1110),and 2273 bp of 3′ untranslated cDNA (corresponding to SEQ ID NO: 1 atnucleotides 1111-3383). The 88C genomic DNA described in Example 1above, corresponds to SEQ ID NO: 1 at nucleotides 424-809.

[0051] The deduced amino acid sequences of 88-2B and 88C revealedhydrophobicity profiles characteristic of GPCRs, including sevenhydrophobic domains corresponding to GPCR transmembrane domains.Sequence comparisons with other GPCRs also revealed a degree ofidentity. Significantly, the deduced amino acid sequences of both 88-2Band 88C had highest identity with the sequences of the chemokinereceptors.

[0052] Table 1 presents the results of these amino acid sequencecomparisons. TABLE 1 Chemokine Receptors 88-2B 88C IL-8RA 30% 30% IL-8RB31% 30% CCCKR1 62% 54% CCCKR2A 46% 66% CCCKR2B 50% 72% 88-2B 100%  50%88-C 50% 100% 

[0053] Table 1 shows that 88-2B is most similar to CCCKR1 (62% identicalat the amino acid level) and 88C is most similar to CCCKR2 (72%identical at the amino acid level).

[0054] The deduced amino acid sequences of 88-2B and 88C also reveal theintracellular and extracellular domains characteristic of GPCRs. The88-2B extracellular domains correspond to the amino acid sequenceprovided in SEQ ID NO:3, and SEQ ID NO:4, at amino acid residues 1-36,93-107, 171-196, and 263-284. The extracellular domains of 88-2B areencoded by polynucleotide sequences corresponding to SEQ ID NO:3 atnucleotides 362-469, 638-682, 872-949, and 1148-1213. Extracellulardomains of 88C include amino acid residues 1-32, 89-112, 166-191, and259-280 in SEQ ID NO: 1 and SEQ ID NO:2. The 88C extracellular domainsare encoded by polynucleotide sequences that correspond to SEQ ID NO: 1at nucleotides 55-150, 319-390, 550-627, and 829-894. The intracellulardomains of 88-2B include amino acids 60-71, 131-151, 219-240, and306-355 of SEQ ID NO:3 and SEQ ID NO:4. Those domains are encoded bypolynucleotide sequences corresponding to SEQ ID NO:3 at nucleotides539-574, 752-814, 1016-1081, and 1277-1426, respectively. The 88Cintracellular domains include amino acid residues 56-67, 125-145,213-235, and 301-352 of SEQ ID NO:1 and SEQ ID NO:2. The intracellulardomains of 88C are encoded by polynucleotide sequences corresponding toSEQ ID NO:1 at nucleotides 220-255, 427-489, 691-759, and 955-1110.

[0055] In addition, a macaque 88C DNA was amplified by PCR from macaquegenomic DNA using primers corresponding to 5′ and 3′ flanking regions ofthe human 88C cDNA. The 5′ primer corresponded to the region immediatelyupstream of and including the initiating Met codon. The 3′ primer wascomplementary to the region immediately downstream of the terminationcodon. The primers included restriction sites for cloning intoexpression vectors. The sequence of the 5′ primer wasGACAAGCTTCACAGGGTGGAACAAGATG (with the HindIII site underlined) (SEQ IDNO: 17) and the sequence of the 3′ primer wasGTCTCTAGACCACTTGAGTCCGTGTCA (with the XbaI site underlined) (SEQ ID NO:18). The conditions of the PCR amplification were 94° C. for eightminutes, then 40 cycles of 94° C. for one minute, 55° C. for 45 seconds,and 72° C. one minute. The amplified products were cloned into theHindIII and XbaI sites of pcDNA3 and a clone was obtained and sequenced.The full length macaque cDNA and deduced amino acid sequences arepresented in SEQ ID NOs:19 and 20, respectively. The nucleotide sequenceof macaque 88C is 98% identical to the human 88C sequence. The deducedamino acid sequences are 97% identical.

Example 3

[0056] The mRNA expression patterns of 88-2B and 88C were determined byNorthern blot analyses.

[0057] Northern blots containing immobilized poly A++ RNA from a varietyof human tissues were purchased from Clontech Laboratories, Inc., PaloAlto, Calif. In particular, the following tissues were examined: heart,brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, colon and peripheralblood leukocytes.

[0058] A probe specific for 88-2B nucleotide sequences was generatedfrom cDNA clone 478. The cDNA insert in clone 478 contains sequencecorresponding to SEQ ID NO: 3 at nucleotides 641-1915. To generate aprobe, clone 478 was digested and the insert DNA fragment was isolatedfollowing gel electrophoresis. The isolated insert fragment was thenradiolabeled with ³²P-labeled nucleotides, using techniques known in theart.

[0059] A probe specific for 88C nucleotide sequences was generated byisolating and radiolabeling the insert DNA fragment found in clone 493.The insert fragment from clone 493 contains sequence corresponding toSEQ ID NO: 1 at nucleotides 421-1359. Again, conventional techniquesinvolving ³²P-labeled nucleotides were used to generate the probe.

[0060] Northern blots probed with 88-2B revealed an approximately 1.8 kbmRNA in peripheral blood leukocytes. The 88C Northerns showed anapproximately 4 kb MRNA in several human tissues, including a strongsignal when probing spleen or thymus tissue and less intense signalswhen analyzing MRNA from peripheral blood leukocytes and smallintestine. A relatively weak signal for 88C was detected in lung tissueand in ovarian tissue.

[0061] The expression of 88C in human T-cells and in hematopoietic celllines was also determined by Northern blot analysis. Levels of 88C inCD4^(+ and CD)8⁺T-cells were very high. The transcript was present atrelatively high levels in myeloid cell lines THP1 and HL-60 and alsofound in the B cell line Jijoye. In addition, the cDNA was a relativelyabundant transcript in a human macrophage cDNA library based on PCRamplification of library subfractions.

Example 4

[0062] The 88-2B and 88C cDNAs were expressed by recombinant methods inmammalian cells.

[0063] For transient transfection experiments, 88C was subcloned intothe mammalian cell expression vector pBJ1 [Ishi et al., J. Biol. Chem270:16435-16440 (1995)]. The construct included sequences encoding aprolactin signal sequence for efficient cell surface expression and aFLAG epitope at the amino terminus of 88C to facilitate detection of theexpressed protein. The FLAG epitope consists of the sequence “DYKDDDD.”COS-7 cells were transiently transfected with the 88C expressionplasmid using Lipofectamine (Life Technology, Inc., Grand Island, N.Y.)following the manufacturer's instructions. Briefly, cells were seeded in24-well plates at a density of 4×10⁴ cells per well and grown overnight.The cells were then washed with PBS, and 0.3 mg of DNA mixed with 1.5 μlof lipofectamine in 0.25 ml of Opti-MEM was added to each well. After 5hours at 37° C., the medium was replaced with medium containing 10% FCS.quantitative ELISA confirmed that 88C was expressed at the cell surfacein transiently transfected COS-7 cells using the M1 antibody specificfor the FLAG epitope (Eastman Co., New Haven, Conn.).

[0064] The FLAG-tagged 88C receptor was also stably transfected intoHEK-293 cells, a human embryonic kidney cell line, using transfectionreagent DOTAP(N-[1-[(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummethylsulfate,Boehringer-Mannheim, Inc., Indianapolis, Ind.) according to themanufacturer's recommendations. Stable lines were selected in thepresence of the drug G418. The transfected HEK-293 cells were evaluatedfor expression of 88C at the cell surface by ELISA, using the Mlantibody to the FLAG epitope. ELISA showed that 88C tagged with the FLAGepitope was expressed at the cell surface of stably transformed HEK-293Cells.

[0065] The 88-2B and 88C cDNAs were used to make stable HEK-293transfectants. The 88-2B receptor cDNA was cloned behind thecytomegalovirus promoter in pRc/CMV (Invitrogen Corp., San Diego,Calif.) using a PCR-based strategy. The template for the PCR reactionwas the cDNA insert in clone 777. The PCR primers were 88-2B-3(containing an internal XbaI site) and 88-2B-5 (containing an internalHindIII site).

[0066] The nucleotide sequence of primer 88-2B-3 is presented in SEQ IDNO:9; the nucleotide sequence of primer 88-2B-5 is presented in SEQ IDNO: 10. An 1104 bp region of cDNA was amplified. Followingamplification, the DNA was digested with XbaI and HindIII and clonedinto similarly digested pRc/CMV. The resulting plasmid was named 777XP2,which contains 18 bp of 5′ untranslated sequence, the entire codingregion of 88-2B, and 3 bp of 3′ untranslated sequence. For the 88Csequence, the full-length cDNA insert in clone 134 was not furthermodified before transfecting HEK-293 cells.

[0067] To create stably transformed cell lines, the pRc/CMV recombinantclones were transfected using transfection reagent DOTAP(N-[1-[(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummethylsulfate,Boehringer-Mannheim, Inc., Indianapolis, Ind.) according to themanufacturer's recommendations, into HEK-293 cells, a human embryonickidney cell line. Stable lines were selected in the presence of the drugG418. Standard screening procedures (i.e., Northern blot analyses) wereperformed to identify stable cell lines expressing the highest levels of88-2B and 88C mRNA.

EXAMPLE 5

[0068] A. Ca++ Flux Assays

[0069] To analyze polypeptide expression, a functional assay forchemokine receptor activity was employed. A common feature of signalingthrough the known chemokine receptors is that signal transduction isassociated with the release of intracellular calcium cations. Therefore,intracellular Ca++ concentration in the transfected HEK-293 cells wasassayed to determine whether the 88-2B or 88C receptors responded to anyof the known chemokines.

[0070] HEK-293 cells, stably transfected with 88-2B, 88C (without theFLAG epitope sequence), or a control coding region (encoding IL8R orCCCKR2, see below) as described above, were grown in T75 flasks toapproximately 90% confluence in MEM+10% serum. Cells were then washed,harvested with versene (0.6 mM EDTA, 10 mM Na₂HPO₄, 0.14 M NaCl, 3 mMKCl, and 1 mM glucose), and incubated in MEM+10% serum+1 μM Fura-2 AM(Molecular Probes, Inc., Eugene, Oreg.) for 30 minutes at roomtemperature. Fura-2 AM is a Ca++-sensitive dye. The cells wereresuspended in Dulbecco's phosphate-buffered saline containing 0.9 mMCaCl₂ and 0.5 mM MgCl₂ (D-PBS) to a concentration of approximately 10⁷cells/ml and changes in fluorescence were monitored using a fluorescencespectrophotometer (Hitachi Model F-4010). Approximately 10⁶ cells weresuspended in 1.8 ml D-PBS in a cuvette maintained at 37° C. Excitationwavelengths alternated between 340 and 380 nm at 4 second intervals; theemission wavelength was 510 nm. Test compositions were added to thecuvette via an injection port; maximal Ca++ flux was measured upon theaddition of ionomycin.

[0071] Positive responses were observed in cells expressing IL-8RA whenstimulated with IL-8 and also when CCCKR2 was stimulated with MCP-1 orMCP-3. However, HEK-293 cells expressing either 88-2B or 88C failed toshow a flux in intracellular Ca++ concentration when exposed to any ofthe following chemokines: MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1μ, IL8,NAP-2, gro/MGSA, IP-10, ENA-78, or PF-4. (Peprotech, Inc., Rocky Hill,N.J.).

[0072] Using a more sensitive assay, a Ca++ flux response to RANTES wasobserved microscopically in Fura-2 AM-loaded cells expressing 88-2B. Theassay involved cells and reagents prepared as described above. RANTES(Regulated on Activation, Normal T Expressed and Secreted) is a CCchemokine that has been identified as a chemoattractant and activator ofeosinophils. See Neote et al., supra. This chemokine also mediates therelease of histamine by basophils and has been shown to function as achemoattractant for memory T cells in vitro. Modulation of 88-2Breceptor activities is therefore contemplated to be useful in modulatingleukocyte activation.

[0073] FLAG tagged 88C receptor was expressed in HEK-293 cells andtested for chemokine interactions in the CA++ flux assay. Cell surfaceexpression of 88C was confirmed by ELISA and by FACScan analysis usingthe M1 antibody. The chemokines RANTES, MIP-1α, and MIP-1β all induced aCa++ flux in 88C-transfected cells when added at a concentration of 100nM.

[0074] Ca++ flux assays can also be designed to identify modulators ofchemokine receptor binding. The preceding fluorimetric or microscopicassays are carried out in the presence of test compounds. If Ca++ fluxis increased in the presence of a test compound, that compound is anactivator of chemokine receptor binding. In contrast, a diminished Ca++flux identifies the test compound as an inhibitor of chemokine receptorbinding.

[0075] B. Phosphoinositol Hydrolysis

[0076] Another assay for ligands or modulators involves monitoringphospholipase C activity, as described in Hung et al., J. Biol. Chem.116:827-832 (1992). Initially, host cells expressing a chemokinereceptor are loaded with ³H-inositol for 24 hours. Test compounds (i.e.,potential ligands) are then added to the cells and incubated at 37° C.for 15 minutes. The cells are then exposed to 20 mM formic acid tosolubilize and extract hydrolyzed metabolites of phosphoinositolmetabolism (i.e., the products of phospholipase C-mediated hydrolysis).The extract is subjected to anion exchange chromatography using an AG1X8anion exchange column (formate form). Inositol phosphates are elutedwith 2 M ammonium formate/0. 1 M formic acid and the ³H associated withthe compounds is determined using liquid scintillationspectrophotometry. The phospholipase C assay can also be exploited toidentify modulators of chemokine receptor activity. The aforementionedassay is performed as described, but with the addition of a potentialmodulator. Elevated levels of detectable label would indicate themodulator is an activator;

[0077] depressed levels of the label would indicate the modulator is aninhibitor of chemokine receptor activity.

[0078] The phospholipase C assay was performed to identify chemokineligands of the FLAG-tagged 88C receptor. Approximately 24 hours aftertransfection, COS-7 cells expressing 88C were labeled for 20-24 hourswith myo-[2-³H]inositol (1 μCi/ml) in inositol-free medium containing10% dialyzed FCS. Labeled cells were washed with inositol-free DMEMcontaining 10 mM LiCl and incubated at 37° C. for 1 hour withinositol-free DMEM containing 10 mM LiCl and one of the followingchemokines: RANTES, MIP-1β, MIP-1α, MCP-1, IL-8, or the murine MCP-1homolog JE. Inositol phosphate (IP) formation was assayed as describedin the previous paragraph. After incubation with chemokines, the mediumwas aspirated and cells were lysed by addition of 0.75 ml of ice-cold 20mM formic acid (30 min). Supernatant fractions were loaded onto AG1-X8Dowex columns (Biorad, Hercules, Calif.), followed by immediate additionof 3 ml of 50 mM NH₄OH. The columns were then washed with 4 ml of 40 mMammonium formate, followed by elution with 2 M ammonium formate. Totalinositol phosphates were quantitated by counting beta-emissions.

[0079] Because it has been shown that some chemokine receptors, such asIL8RA AND IL8RB, require contransfection with an exogenous G proteinbefore signaling can be detected in COS-7 cells, the 88C receptor wasco-expressed with the chimeric G protein Gqi5 (Conklin, et al., Nature363:274-276, (1993). Gqi5 ia a G protein which has the carboxyl terminalfive amino acids of Gi (which bind to the receptor) spliced onto Gαq.Co-transfection with Gqi5 significantly potentiates signaling by CCCKR1and CCKR2B. Co-transfection with Gqi5 revealed that 88C signaled well inresponse to RANTES, MIP-1β, and MIP-1α, but not in response to MCP-1,IL-8 or the murine MCP-1 homologue JE. Dose-response curves revealedEC₅₀ values of 1 nM for RANTES, 6 nM for MIP-1β, and 22 nM for MIP-1 α.

[0080] 88C is the first cloned human receptor with a signaling responseto MIP-1β. Compared with other CC chemokines, MIP-1βclearly has a uniquecellular activation pattern. It appears to activate T cells but notmonocytes (Baggiolini et al., supra) which is consistent with receptorstimulation studies. For example, while MIP-1βbinds to CCCKR1, it doesnot induce calcium flux (Neote et al., supra). In contrast, MIP-1α andRANTES bind to and causes signaling in CCCKR1 and CCCKR5 (RANTES alsocauses activation of CCCKR3). MIP-1p thus appears to be much moreselective than other chemokines of the CC chemokine family. Suchselectivity is of therapeutic significance because a specific beneficialactivity can be stimulated (such as suppression of HIV infection)without stimulating multiple leukocyte populations which results ingeneral pro-inflammatory activities.

[0081] C. Binding Assays

[0082] Another assay for receptor interaction with chemokines was amodification of the binding assay described by Ernst et al., J. Immunol.152:3541-3549 (1994). MIP-1β as labeled using the Bolton and Hunterreagent (di-iodide, NEN, Wilmington, Del.), according to themanufacturer's instructions. Unconjugated iodide was separated fromlabeled protein by elution using a PD-10 column (Pharmacia) equilibratedwith PBS and BSA (1% w/v). The specific activity was typically 2200Ci/mmole. Equilibrium binding was performed by adding ¹²⁵I-labeledligand with or without a 100-fold excess of unlabeled ligand, to 5×10⁵HEK-293 cells transfected with 88C tagged with the FLAG epitope inpolypropylene tubes in a total volume of 300 μl(50 mM HEPES pH 7.4, 1 mMCaCl₂, MgCl₂, 0.5% BSA) and incubating for 90 minutes at 27° C. withshaking at 150 rpm. The cells were collected, using a Skatron cellharvester (Skatron Instruments Inc., Sterling, Va.), on glass fiberfilters presoaked in 0.3% polyethyleneimine and 0.2% BSA. After washing,the filters were removed and bound ligand was quantitated by countinggamma emissions. Ligand binding by competition with unlabeled ligand wasdetermined by incubation of 5×10⁵ transfected cells (as above) with 1.5nM of radiolabeled ligand and the indicated concentrations of unlabeledligand. The samples were collected, washed and counted as above. Thedata was analyzed using the curve-fitting program Prism (GraphPad Inc.,San Diego, Calif.) and the iterative non-linear regression program,LIGAND (PM220).

[0083] In equilibrium binding assays, 88C receptor bound radiolabeledMIP-1β in a specific and saturable manner. Analysis of this binding databy the method of Scatchard revealed a dissociation constant (Kd) of 1.6nM. Competition binding assays using labeled MIP-1β revealedhigh-affmity binding of MIP-1β(IC₅₀=7.4 nM), RANTES (IC₅₀=6.9 nM), andMIP-1α(IC_(50=7.4) nM), consistent with the signaling data obtained intransiently transfected COS-7 cells as discussed in section B above.

[0084] Example 6

[0085] The chemokines MIP-1α, MIP-1β and RANTES have been shown toinhibit replication of HIV-1 and HIV-2 in human peripheral bloodmononuclear cells and PM1 cells (Cocchi et al., supra). In view of thisfinding and in view of the results described in Example 5, the presentinvention contemplates that activation of or ligand binding to the 88Creceptor may provide a protective role in HIV infection.

[0086] Recently, it has been reported that the orphan G protein-coupledreceptor, fusin, can act as a co-receptor for HIV entry. Fusin/CXCR4 incombination with CD4, the primary HIV receptor, apparently facilitatesHIV infection of cultured T cells ([Feng et al., Science 272:872-877(1996)]. Based upon the homology of fusin to chemokine receptors and thechemokine binding profile of 88C, and because 88C is constitutivelyexpressed in T cells and abundantly expressed in macrophages, 88C islikely to be involved in viral and HIV infection.

[0087] The function of 88C and 88-2B as co-receptors for HIV wasdetermined by transfecting cells which express CD4 with 88C or 88-2B andchallenging the co-transfected cells with HIV. Only cells expressingboth CD4 and a functional co-receptor for HIV become infected. HIVinfection can be determined by several methods. ELISAs which test forexpression of HIV antigens are commercially available, for exampleCoulter HIV-1 _(p)24 antigen assay (U.S. Pat. No. 4,886,742), CoulterCorp., 11800 SW 147th Ave., Miami, Fla. 33196. Alternatively, the testcells can be engineered to express a reporter gene such as LACZ attachedto the HIV LTR promoter [Kimpton et al., J. Virol. 66:2232-2239 (1992)].In this method, cells that are infected with HIV are detected by acolorimetric assay.

[0088] 88C was transiently transfected into a cat cell line, CCC[Clapham, et al., 181:703-715 (1991)], which had been stably tranformedto express human CD4 (CCC-CD4). These cells are normally resistant toinfection by any strain of HIV-1 because they do not endogenouslyexpress 88C. In these experiments, CCC/CD4 cells were transientlytransfected with 88C cloned into the expression vector pcDNA3.1(Invitrogen Corp., San Diego, Calif.) using lipofectamine (Gibco BRL,Gaithersburg, Md.). Two days after transfection, cells were challengedwith HIV. After 4 days of incubation, cells were fixed and stained forp24 antigen as a measure of HIV infection. 88C expression by these cellsrendered them susceptible to infection by several strains of HIV-1.These strains included four primary non-syncytium-inducing HIV-1isolates (M23, E80, SL-2 and SF-162) which were shown to use only 88C asa co-receptor but not fusin. Several primary syncytium-inducing strainsof HIV-1 (2006, M13, 2028 and 2076) used either 88C or fusin as aco-receptor. Also, two established clonal HIV-1 viruses (GUN-1 and 89.6)used either 88C or fusin as a co-receptor.

[0089] It has been reported that some strains of HIV-2 can infectcertain CD4-negative cell lines, thus implying a direct interaction ofHIV-2 with a receptor other than CD4 [Clapham et al., J. Virol.66:3531-3537 (1992)] For some strains of HIV-2, this infection isfacilitated by the presence of soluble CD4 (sCD4). Since 88-2B shareshigh sequence similarity with other chemokine receptors that act as HIVco-receptors (namely 88C and fusin), 88-2B was considered to be a likelyHIV-2 co-receptor. The role of 88-2B as an HIV-2 co-receptor wasdemonstrated using HIV-2 strain ROD/B. Cat CCC cells which do notendogenously express CD4 were transfected with 88-2B. In theseexperiments, cells were transfected with pcDNA3.1 containing 88-2B usinglipofectamine and infected with HIV-2 48 hours later. Three days afterinfection, cells were immunostained for the presence of HIV-2 envelopeglycoproteins. The presence of sCD4 during HIV-2_(ROD/B) challengeincreased the infection of these cells by 10-fold. The entry of HIV-2into the 88-2B transfected cells could be blocked by the presence of400-800 ng/ml eotaxin, one of the ligands for 88-2B. The baselineinfectivity levels of CCC/88-2B (with no soluble CD4) were equivalent toCCC cells which were not transfected with 88-2B.

[0090] The role of 88-2B and 88C as co-receptors for HIV was confirmedby preparing and challenging cell lines stably transformed to express88C or 88-2B with various strains of HIV and SIV. These results aredescribed in Example 7.

[0091] Alternatively, the co-receptor role of 88C and 88-2B can bedemonstrated by an experimental method which does not require the use oflive virus. In this method, cell lines co-expressing 88C or 88-2B, CD4and a LACZ reporter gene are mixed with a cell line co-expressing theHIV envelope glycoprotein (ENV) and a transcription factor for thereporter gene construct [Nussbaum et al., J. Virol. 68:5411 (1994)].Cells expressing a functional co-receptor for HIV will fuse with the ENVexpressing cells and thereby allow expression of the reporter gene. Inthis method, detection of reporter gene product by colorimetric assayindicates that 88C or 88-2B function as a co-receptor for HIV.

[0092] The mechanism by which chemokines inhibit viral infection has notyet been elucidated. One possible mechanism involves activation of thereceptor by binding of a chemokine. The binding of the chemokine leadsto signal transduction events in the cell that renders the cellresistant to viral infection and/or prevents replication of the virus inthe cell. Similar to interferon induction, the cell may differentiatesuch that it is resistant to viral infection, or an antiviral state isestablished. Alternatively, a second mechanism involves directinterference with viral entry into cells by blocking access of viralenvelope glycoproteins to the co-receptor by chemokine binding. In thismechanism, G-protein signaling is not required for chemokine suppressionof HIV infection.

[0093] To distinguish between two mechanisms by which 88C or 88-2B mayfunction as co-receptors for viral or HIV infection, chemokine bindingto the receptor is uncoupled from signal transduction and the effect ofthe chemokine on suppression of viral infection is determined.

[0094] Ligand binding can be uncoupled from signal transduction by theaddition of compounds which inhibit G-protein mediated signaling. Thesecompounds include, for example, pertussis toxin and cholera toxin. Inaddition, downstream effector polypeptides can be inhibited by othercompounds such as wortmannin. If G-protein signaling is involved insuppression of viral infection, the addition of such compounds wouldprevent suppression of viral infection by the chemokine. Alternatively,key residues or receptor domains of 88C or 88-2B receptor required forG-protein coupling can be altered or deleted such that G-proteincoupling is altered or destroyed but chemokine binding is not affected.

[0095] Under these conditions, if chemokines are unable to suppressviral or HIV infection, then signaling through a G-protein is requiredfor suppression of viral or HIV infection. If however, chemokines areable to suppress viral infection, then G-protein signaling is notrequired for chemokine suppression of viral infection and the protectiveeffects of chemokines may be due to the chemokine blocking theavailability of the receptor for the virus.

[0096] Another approach involves the use of antibodies directed against88C or 88-2B. Antibodies which bind to 88C or 88-2B which can be shownnot to elicit G-protein signaling may block access to the chemokine orviral binding site of the receptor. If in the presence of antibodies to88C or 88-2B, viral infection is suppressed, then the mechanism of theprotective effects of chemokines is blocking viral access to itsreceptor. Feng et al. (1996) reported that antibodies to the aminoterminus of the fusin receptor suppressed HIV infection.

[0097] Example 7

[0098] Cell lines were stably transformed with 88C or 88-2B to furtherdelineate the role of 88C and 88-2B in HIV infection. Kimpton andEmerman [“Detection of Replication-Competent and Pseudotyped HumanImmunodeficiency Virus with a Sensitive Cell Line on the Basis ofActivation of an Integrated Beta-Galactosidase Gene,” J. Virol,66(4):2232-2239 (1992)] previously described an indicator cell line,herein identified as HeLa-MAGI cells. HeLa-MAGI cells are HeLa cellsthat have been stably transformed to express CD4 as well as integratedHIV-1 LTR which drives expression of a nuclear localized β-galactosidasegene. Integration of an HIV provirus in the cells leads to production ofthe viral transactivator, Tat, which then turns on expression of theβ-galactosidase gene. The number of cells that stain positive with X-galfor β-galactosidase activity in situ is directly proportional to thenumber of infected cells.

[0099] These HeLa-MAGI cells can detect lab-adapted isolates of HIV-1but only a minority of primary isolates [Kimpton and Emerman, supra],and cannot detect most SIV isolates [Chackerian et al.,“Characterization of a CD4-Expressing Macaque Cell Line that can DetectVirus After A Single Replication Cycle and can be infected by DiverseSimian Immunodeficiency Virus Isolates,” Virology, 213(2):6499-6505(1995)].

[0100] In addition, Harrington and Geballe [“Co-Factor Requirement forHuman Immunodeficiency Virus Type 1 Entry into a CD4-Expressing HumanCell Line, J. Virol., 67:5939-5947 (1993)] described a cell line basedon U373 cells that had been engineered to express CD4 and the sameLTR-β-galactosidase construct. It was previously shown that this cellline, herein identified as U373-MAGI, could not be infected with any HIV(M or T-tropic) strain of HIV, but could be rendered susceptible toinfection by fusion with HeLa cells (Harrington and Geballe, supra).

[0101] In order to construct indicator cell lines that could detecteither macrophage or T cell tropic viruses, epitope-tagged 88C or 88-2Bencoding DNA was transfected into HeLa-MAGI or U373-MAGI cells byinfection with a retroviral vector to generate HeLa-MAGI-88C orU373-MAGI-88C cell lines, respectively. Expression of the co-receptorson the cell surface was demonstrated by immunostaining live cells usingthe anti-FLAG M1 antibody and by RT-PCR.

[0102] The 88C and 88-2B genes utilized to construct HeLa-MAGI-88C andU373-MAGI-88C included sequences encoding the prolactin signal peptidefollowed by a FLAG epitope as described in Example 4. This gene wasinserted into the retroviral vector pBabe-Puro [Morgenstern and Land,Nucleic Acids Research, 18(12):3587-3596 (1990)].

[0103] High titer retroviral vector stocks pseudotyped with the VSV-Gprotein were made by transient transfection as described in Bartx etal., J. Virol. 70:2324-2331 (1996), and used to infect HeLa-MAGI andU373-MAGI cells. Cells resistant to 0.6 μ/ml puromycin (HeLa) or 1 μ/mlpuromycin (U373) were pooled. Each pool contained at least 1000independent transduction events. An early passage (passage 2) stock ofthe original HeLa-MAGI cells (Kimpton and Emerman, supra) was used tocreate HeLa-MAGI-88C cells.

[0104] Infections of the indicator cell lines with HIV were performed in12-well plates with 10-fold serial dilutions of 300 μl of virus in thepresence of 30 μ/ml DEAE-Dextran as described (Kimpton and Emerman,supra).

[0105] All HIV-1 strains and SIV_(mac 239) were all obtained from theNIH AIDS Reference and Reagent Program. Molecular clones of primaryHIV-2_(7312A) [Gao et al., “Genetic Diversity of Human ImmunodeficiencyVirus Type 2: Evidence for Distinct Sequence Subtypes with Differencesin Virus Biology,” J. Virol., 68(11):7433-7447 (1992)] and SIVsmPbj1.9[Dewhurst et al., “Sequence Analysis and Acute Pathogenicity ofMolecularly Cloned SIV_(smm)-PBj14,”Nature, 345:636-640 (1990)] wereobtained from B.

[0106] Hahn (UAB). All other SIV_(mne)isolates were obtained from JulieOverbaugh (U.

[0107] Washington, Seattle). Stocks from cloned proviruses were made bytransient transfection of 293 cells. Other viral stocks were made bypassage of virus in human peripheral blood mononuclear cells or inCEMx174 cells (for SIV stocks.) Viral stocks were normalized by ELISA orp24^(gag) (Coulter Immunology) or p27^(gag) (Coulter Immunology) forHIV-1 and HIV-2/SIV, respectively, using standards provided by themanufacturer.

[0108] U373-MAGI-88C cells and U373-MAGI cells (controls) and wereinfected with limiting dilutions of a T-tropic strain of HIV-1(HIV_(LAI)), an M-tropic strain (HIV_(YU-2)), and an SIV isolate,SIV_(MAC)239.Infectivity was measured by counting the number of bluecells per well per volume of virus (Table 2). TABLE 2 titer on cell line(IU/ml)^(b) virus strain^(a) U373-MAGI U373-MAGI-88C HIV-1_(LAI) <100<100 HIV-1_(YU-2) <100 2.2 × 10⁶ SIV_(MAC)239 1.2 × 10³   4 × 10⁵

[0109] Two days after infection, cells were fixed and stained forβ-galactosidase activity with X-gal. The U373-derived MAGI cells werestained for 120 minutes at 370° C. and the HeLa-derived MAGI cells werestained for 50 minutes at 37° C. Background staining of non-infectedcells never exceeded more than approximately three blue cells per well.Only dark blue cells were counted, and syncytium with multiple nucleiwere counted as a single infected cell. The infectious titer is thenumber of blue cells per well multiplied by the dilution of virus andnormalized to 1 ml. The titer of HIV_(YU-2) on U373-MAGI-88C cells was2×10^(6.) In contrast, the titer of HIV-1_(LA1), was less than 100 onU373-MAGI-88C. Thus, the specificity of a particular HIV strain for 88Cvaried by four orders of magnitude.

[0110] Although SIV_(MAC)239 infection was increased to 4×10⁵ inU373-MAGI-88C it also clearly infected U373-MAGI cells (Table 2).

[0111] Next, a series of primary uncloned HIV strains and clonedM-tropic strains of HIV-1 were analyzed for their ability to infectindicator cell lines that express 88C.

[0112] As described above, HeLa-MAGI and HeLa-MAGI-88C cells wereinfected with limiting dilutions of various HIV strains. The two clonedM-tropic viruses, HIV_(JR-CSF) and HIV_(YU-2), both infectedHeLa-MAGI-88C, but not HeLa-MAGI cells, showing that both strains use88C as a co-receptor (Table 3, See note c). However, a great disparityin the ability of each of these two viral strains to infectHeLa-MAGI-88C cells was observed, 6.2×10⁵ IU/ml for HIV_(YU-2) and1.2×10⁴ for HIV_(JR-CSF). The infectivity of virus stock (Table 3) isthe number of infectious units per physical particle (represented hereby the amount of viral core protein). In addition, it was observed thatthe infectivity of these two cloned viral strains differed by over50-fold in viral stocks that were independently prepared.

[0113] The variability of infectivity of primary viral isolates wasfurther examined by analyzing a collection of twelve different unclonedvirus stocks from three different clades (Table 3). Three clade Aprimary isolates, three clade E isolates, and three additional clade Bisolates from geographically diverse origins were used. With all ninestrains, the primary strains of HIV could be detected on HeLa-MAGI-88Ccells, but not on HeLa-MAGI cells (Table 3). However, the efficacy ofinfection varied from five infectious units per ng p24^(gag) to over 100infectious units per ng p24^(gag) (table 3). These results indicate thatabsolute infectivity of M-tropic strains varies considerably and isindependent of clade. A hypothesis that may explain this discrepancy mayinvolve the affinity of the V3 loop of each viral strain for 88C afterCD4 binding [Trkola et al., Nature, 384(6605):184-187 (1996); Wu et al.,Nature, 384(6605):179-183 (1996)].

[0114] Table 3 TABLE 3 viral sub-type titer (IU/ml) (country of on HeLa-P24^(gag) virus strain^(a) origin)^(b) MAGI-88C^(c) ng/mlInfectivity^(d) HIV-1_(YU-2) B (USA) 6.2 × 10⁵ 2200 281 HIV-1_(JR-CSF) B(USA) 12000  2800 4.2 HIV-1_(TH020) E (Thailand) 4133 93 44HIV-1_(TH021) E (Thailand) 4967 52 96 HIV-1_(TH022) E (Thailand)  200 1513 HIV-1_(US660) B (USA) 2367 127 19 HIV-1_(UG031) A (Uganda) 1633 71 23HIV-1_(RW009) A (Rwanda) 3333 158 21 HIV-1_(RW026) A (Rwanda)  739 1435.2 HIV-1_(US727) B (USA) 14,067   289 49 HIV-1_(US056) B (USA) 5833 28421 HIV-1_(LAI) B (France) 2.8 × 10⁵ 167 1600

[0115] The ability of the HeLa-MAGI-88C cells to detect HIV-2 and otherSIV strains was also determined. HIV-2_(Rod) has been reported to usefusin as a receptor even in the absence of CD4 [Endres et al., Cell,87(4):745-756 (1996)]. HIV-2_(Rod) is able to infect HeLa-MAGI cells,however its infectivity is enhanced at least 10-fold in HeLa-MagI-88C(Table 4). HeLa cells endogenously express fusin. Thus, the molecularclone of HIV-2_(Rod) is dual tropic, and is able to use 88C as one ofits co-receptors in addition to CXCR4. Similarly, a primary strain ofHIV-2_(7312A) infected HeLa-MAGI-88C cells and not the HeLa-MAGI cells,indicating that like primary strain of HIV-1, it uses 88C as a receptor.TABLE 4 titer (IU/ml) titer (IU/ml) Infectivity on HeLa- on HeLa- onHeLa- virus strain^(a) reference MAGI^(b) MAGI-88C MAGI-88C^(c)HIV-2_(ROD9) (Guyader et al., 967 5900 13 1987) HIV-2_(7312A) (Gao etal., <30 6500 17 1994) SIV_(MAC)239 (Naidu et al., <30 20900  90 1988)SIV_(MNE)c18 (Overbaugh et <30 15700  19 al., 1991) SIV_(MNE)170(Rudensey et <30 10700  27 al., 1995) SIV_(SM)Pbj1.9 (Dewhurst et <30 776 ND^(d) al., 1990) SIV_(AGM)9063 (Hirsch et al., <30  50 <1 1995)

[0116] None of the SIV strains tested infected the HeLa-MAGI cells(Table 4), and none infected HeLa-MAGI cells that expresses 88-2B. Thisindicates that an alternative co-receptor used by SIV in U373 cells isnot expressed in HeLa cells, and is not 88-2B. All SIV strains testedinfected the HeLa-MAGI-88C cells to some extent (Table 3) indicatingthat all of the tested SIV strains use at least 88C as one of theirco-receptors.

[0117] The classification of M-tropic and T-tropic strains of HIV in thepast has often been correlated with another designation “non-syncytiuminducing” (NSI), and “syncytium inducing” (SI), respectively. Assaysbased on the cell lines described herein are sensitive to syncytiumformation. The infected cells can form large and small foci of infectioncontaining multiple nuclei (Kimpton and Emerman, supra).

[0118] Experiments using multiple different viral strains andU373-MAGI-88C or HeLa-MAGI-88C indicate that SI/NSI designation is notmeaningful because all viral strains formed syncytia if the correctco-receptor was present. These experiments show that syncytium formationis more likely a marker for the presence of an appropriate co-receptoron the infected cell, rather than an indication of tropism. Infection ofthe HeLa-MAGI-88C cells with SIV strains reported in the literature tobe non-syncytium forming strains, in particular, SIV_(MAC)239,SIV_(MNE)c18, and SIV_(MNE)170, was remarkable because the size of thesyncytia induced in the monolayer was much larger than those induced byany other the HIV strains.

[0119] EXAMPLE 8

[0120] Mouse monoclonal antibodies which specifically recognize 88C wereprepared. The antibodies were produced by immunizing mice with a peptidecorresponding to the amino terminal twenty amino acids of 88C. Thepeptide was conjugated to Keyhole Limpet Cyanin (KLH) according to themanufacturer's directions (Pierce, Imject maleimide activated KLH),emulsified in complete Freund's adjuvant and injected into five mice.Two additional injections of conjugated peptide in incomplete Freund'sadjuvant occurred at three week intervals. Ten days after the finalinjection, serum from each of the five mice was tested forimmunoreactivity with the twenty amino acid peptide by ELISA. Inaddition, the immunoreactivity of the sera were tested against intact88C receptor expressed on the surface of 293 cells by fluorescenceactivated cell sorting (FACS). The mouse with the best anti-88C activitywas chosen for spleen cell fusion and production of monoclonalantibodies by standard laboratory methods. Five monoclonal cell lines(227K, 227M, 227N, 227P, and 227R) were established which producedantibodies that recognized the peptide by ELISA and the 88C protein on293 cells by FACS. Each antibody was shown to react only with88C-expressing 293 cells, but not with 293 cells expressing the closelyrelated MCP receptor (CCCKR-2). Each antibody was also shown torecognize 88C expressed transiently in COS cells.

[0121] Rabbit polyclonal antibodies were also generated against 88C. Tworabbits were injected with conjugated amino-terminal peptide asdescribed above. The rabbits were further immunized by four additionalinjections of the conjugated amino-terminal peptide. Serum from each ofthe rabbits (2337J and 2470J) was tested by FACS of 293 cells expressing88C. The sera specifically recognized 88C on the surface of 293 cells.

[0122] The five anti-88C monoclonal antibodies were tested for theirability to block infection of cells by SIV, the simian immunodeficiencyvirus closely related to HIV [Lehner et al., Nature Medicine, 2:767(1996)]. Simian CD4⁺T cells, which are normally susceptible to infectionby SIV, were incubated with the SIV_(mac)32HJ5 clone in the presence ofthe anti-88C monoclonal antibody supernatants diluted 1:5. SIV infectionwas measured by determining reverse transcriptase (RT) activity on daynine using the RT detection and quantification method (Quan-T-RT assaykit, Amersham, Arlington Heights, Ill.). Four of the antibodies wereable to block SIV infection: antibody 227K blocked by 53%, 227M by 59%,227N by 47% and 227P by 81%. Antibody 227R did not block SIV infection.

[0123] The five monoclonal antibodies raised against human 88Camino-terminal peptide were also tested for reactivity against macaque88C (SEQ ID NO: 20) (which has two amino acid differences from human 88Cwithin the amino-terminal peptide region).

[0124] The coding regions of human 88C and macaque 88C were cloned intothe expression vector pcDNA3 (Invitrogen). These expression plasmidswere used to transfect COS cells using DEAE. The empty vector was usedas a negative control. Three days after transfection, cells wereharvested and incubated with the five anti-88C monoclonal antibodies andprepared for FACS. The results showed that four of the five antibodies(227K, 227M, 227N, and 227P) recognized macaque 88C while one (227R) didnot. All five antibodies recognized the transfected human 88C, and nonecross-reacted with cells transfected with vector alone. On Feb. 4, 1997,the Applicants deposited hybridoma cell lines 227P, 227R, and 227M withthe American Type Culture Collection (ATCC), which is located at 10801University Blvd., Manassas, Va. 20110-2209, USA, pursuant to theprovisions of the Budapest Treaty. These hybridoma cell lines wereaccorded ATCC designations HB-12281, HB-12282, and HB-12283,respectively.

EXAMPLE 9

[0125] Additional methods may be used to identify ligands and modulatorsof the chemokine receptors of the invention.

[0126] In one embodiment, the invention comprehends a direct assay forligands.

[0127] Detectably labeled test compounds are exposed to membranepreparations presenting chemokine receptors in a functionalconformation. For example, HEK-293 cells, or tissue culture cells, aretransfected with an expression vehicle encoding a chemokine receptor. Amembrane preparation is then made from the transfected cells expressingthe chemokine receptor. The membrane preparation is exposed to¹²⁵I-labeled test compounds (e.g., chemokines) and incubated undersuitable conditions (e.g., 10 minutes at 37° C.). The membranes, withany bound test compounds, are then collected on a filter by vacuumfiltration and washed to remove unbound test compounds. Theradioactivity associated with the bound test compound is thenquantitated by subjecting the filters to liquid scintillationspectrophotometry. The specificity of test compound binding may beconfirmed by repeating the assay in the presence of increasingquantities of unlabeled test compound and noting the level ofcompetition for binding to the receptor. These binding assays can alsoidentify modulators of chemokine receptor binding. The previouslydescribed binding assay may be performed with the followingmodifications. In addition to detectably labeled test compound, apotential modulator is exposed to the membrane preparation. An increasedlevel of membrane-associated label indicates the potential modulator isan activator; a decreased level of membrane-associated label indicatesthe potential modulator is an inhibitor of chemokine receptor binding.

[0128] In another embodiment, the invention comprehends indirect assaysfor identifying receptor ligands that exploit the coupling of chemokinereceptors to G proteins. As reviewed in Linder et al., Sci. Am.,267:56-65 (1992), during signal transduction, an activated receptorinteracts with a G protein, in turn activating the G protein. The Gprotein is activated by exchanging GDP for GTP. Subsequent hydrolysis ofthe G protein-bound GTP deactivates the G protein. One assay for Gprotein activity therefore monitors the release of ³²p_(i) from[γ-³²P]-GTP. For example, approximately 5×10⁷ HEK-293 cells harboringplasmids of the invention are grown in MEM+10% FCS. The growth medium issupplemented with 5 mCi/ml [³²P]-sodium phosphate for 2 hours touniformly label nucleotide pools. The cells are subsequently washed in alow-phosphate isotonic buffer. One aliquot of washed cells is thenexposed to a test compound while a second aliquot of cells is treatedsimilarly, but without exposure to the test compound. Following anincubation period (e.g., 10 minutes), cells are pelleted, lysed andnucleotide compounds fractionated using thin layer chromatographydeveloped with 1 M LiCl. Labeled GTP and GDP are identified byco-developing known standards. The labeled GTP and GDP are thenquantitated by autoradiographic techniques that are standard in the art.Relatively high levels of ³²P-labeled GDP identify test compounds asligands. This type of GTP hydrolysis assay is also useful for theidentification of modulators of chemokine receptor binding.

[0129] The aforementioned assay is performed in the presence of apotential modulator. An intensified signal resulting from a relativeincrease in GTP hydrolysis, producing 32P-labeled GDP, indicates arelative increase in receptor activity. The intensified signal thereforeidentifies the potential modulator as an activator. Conversely, adiminished relative signal for ³²P-labeled GDP, indicative of decreasedreceptor activity, identifies the potential modulator as an inhibitor ofchemokine receptor binding.

[0130] The activities of G protein effector molecules (e.g., adenylylcyclase, phospholipase C, ion channels, and phosphodiesterases) are alsoamenable to assay.

[0131] Assays for the activities of these effector molecules have beenpreviously described. For example, adenylyl cyclase, which catalyzes thesynthesis of cyclic adenosine monophosphate (cAMP), is activated by Gproteins. Therefore, ligand binding to a chemokine receptor thatactivates a G protein, which in turn activates adenylyl cyclase, can bedetected by monitoring cAMP levels in a recombinant host cell of theinvention. Implementing appropriate controls understood in the art, anelevated level of intracellular cAMP can be attributed to aligand-induced increase in receptor activity, thereby identifying aligand. Again using controls understood in the art, a relative reductionin the concentration of cAMP would indirectly identify an inhibitor ofreceptor activity. The concentration of cAMP can be measured by acommercial enzyme immunoassay. For example, the BioTrak Kit providesreagents for a competitive immunoassay (Amersham, Inc., ArlingtonHeights, Ill.). Using this kit according to the manufacturer'srecommendations, a reaction is designed that involves competingunlabeled cAMP with cAMP conjugated to horseradish peroxidase. Theunlabeled cAMP may be obtained, for example, from activated cellsexpressing the chemokine receptors of the invention. The two compoundscompete for binding to an immobilized anti-cAMP antibody. After thecompetition reaction, the immobilized horseradish peroxidase-cAMPconjugate is quantitated by enzyme assay using atetramethylbenzidine/H₂O₂ single-pot substrate with detection of coloredreaction products occurring at 450 nM. The results provide a basis forcalculating the level of unlabeled cAMP, using techniques that arestandard in the art. In addition to identifying ligands binding tochemokine receptors, the cAMP assay can also be used to identifymodulators of chemokine receptor binding. Using recombinant host cellsof the invention, the assay is performed as previously described, withthe addition of a potential modulator of chemokine receptor activity. Byusing controls that are understood in the art, a relative increase ordecrease in intracellular cAMP levels reflects the activation orinhibition of adenylyl cyclase activity. The level of adenylyl cyclaseactivity, in turn, reflects the relative activity of the chemokinereceptor of interest. A relatively elevated level of chemokine receptoractivity identifies an activator; a relatively reduced level of receptoractivity identifies an inhibitor of chemokine receptor activity.

[0132] While the present invention has been described in terms ofspecific embodiments, it is understood that variations and modificationswill occur to those skilled in the art. Accordingly, only suchlimitations as appear in the appended claims should be placed on theinvention.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:20 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 3383 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 55..1110 (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88C polynucleotide and aminoacid sequences” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AGAAGAGCTGAGACATCCGT TCCCCTACAA GAAACTCTCC CCGGGTGGAA CAAG ATG 57 Met 1 GAT TATCAA GTG TCA AGT CCA ATC TAT GAC ATC AAT TAT TAT ACA TCG 105 Asp Tyr GlnVal Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr Ser 5 10 15 GAG CCC TGCCAA AAA ATC AAT GTG AAG CAA ATC GCA GCC CGC CTC CTG 153 Glu Pro Cys GlnLys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu Leu 20 25 30 CCT CCG CTC TACTCA CTG GTG TTC ATC TTT GGT TTT GTG GGC AAC ATG 201 Pro Pro Leu Tyr SerLeu Val Phe Ile Phe Gly Phe Val Gly Asn Met 35 40 45 CTG GTC ATC CTC ATCCTG ATA AAC TGC AAA AGG CTG AAG AGC ATG ACT 249 Leu Val Ile Leu Ile LeuIle Asn Cys Lys Arg Leu Lys Ser Met Thr 50 55 60 65 GAC ATC TAC CTG CTCAAC CTG GCC ATC TCT GAC CTG TTT TTC CTT CTT 297 Asp Ile Tyr Leu Leu AsnLeu Ala Ile Ser Asp Leu Phe Phe Leu Leu 70 75 80 ACT GTC CCC TTC TGG GCTCAC TAT GCT GCC GCC CAG TGG GAC TTT GGA 345 Thr Val Pro Phe Trp Ala HisTyr Ala Ala Ala Gln Trp Asp Phe Gly 85 90 95 AAT ACA ATG TGT CAA CTC TTGACA GGG CTC TAT TTT ATA GGC TTC TTC 393 Asn Thr Met Cys Gln Leu Leu ThrGly Leu Tyr Phe Ile Gly Phe Phe 100 105 110 TCT GGA ATC TTC TTC ATC ATCCTC CTG ACA ATC GAT AGG TAC CTG GCT 441 Ser Gly Ile Phe Phe Ile Ile LeuLeu Thr Ile Asp Arg Tyr Leu Ala 115 120 125 GTC GTC CAT GCT GTG TTT GCTTTA AAA GCC AGG ACG GTC ACC TTT GGG 489 Val Val His Ala Val Phe Ala LeuLys Ala Arg Thr Val Thr Phe Gly 130 135 140 145 GTG GTG ACA AGT GTG ATCACT TGG GTG GTG GCT GTG TTT GCG TCT CTC 537 Val Val Thr Ser Val Ile ThrTrp Val Val Ala Val Phe Ala Ser Leu 150 155 160 CCA GGA ATC ATC TTT ACCAGA TCT CAA AAA GAA GGT CTT CAT TAC ACC 585 Pro Gly Ile Ile Phe Thr ArgSer Gln Lys Glu Gly Leu His Tyr Thr 165 170 175 TGC AGC TCT CAT TTT CCATAC AGT CAG TAT CAA TTC TGG AAG AAT TTC 633 Cys Ser Ser His Phe Pro TyrSer Gln Tyr Gln Phe Trp Lys Asn Phe 180 185 190 CAG ACA TTA AAG ATA GTCATC TTG GGG CTG GTC CTG CCG CTG CTT GTC 681 Gln Thr Leu Lys Ile Val IleLeu Gly Leu Val Leu Pro Leu Leu Val 195 200 205 ATG GTC ATC TGC TAC TCGGGA ATC CTA AAA ACT CTG CTT CGG TGT CGA 729 Met Val Ile Cys Tyr Ser GlyIle Leu Lys Thr Leu Leu Arg Cys Arg 210 215 220 225 AAT GAG AAG AAG AGGCAC AGG GCT GTG AGG CTT ATC TTC ACC ATC ATG 777 Asn Glu Lys Lys Arg HisArg Ala Val Arg Leu Ile Phe Thr Ile Met 230 235 240 ATT GTT TAT TTT CTCTTC TGG GCT CCC TAC AAC ATT GTC CTT CTC CTG 825 Ile Val Tyr Phe Leu PheTrp Ala Pro Tyr Asn Ile Val Leu Leu Leu 245 250 255 AAC ACC TTC CAG GAATTC TTT GGC CTG AAT AAT TGC AGT AGC TCT AAC 873 Asn Thr Phe Gln Glu PhePhe Gly Leu Asn Asn Cys Ser Ser Ser Asn 260 265 270 AGG TTG GAC CAA GCTATG CAG GTG ACA GAG ACT CTT GGG ATG ACG CAC 921 Arg Leu Asp Gln Ala MetGln Val Thr Glu Thr Leu Gly Met Thr His 275 280 285 TGC TGC ATC AAC CCCATC ATC TAT GCC TTT GTC GGG GAG AAG TTC AGA 969 Cys Cys Ile Asn Pro IleIle Tyr Ala Phe Val Gly Glu Lys Phe Arg 290 295 300 305 AAC TAC CTC TTAGTC TTC TTC CAA AAG CAC ATT GCC AAA CGC TTC TGC 1017 Asn Tyr Leu Leu ValPhe Phe Gln Lys His Ile Ala Lys Arg Phe Cys 310 315 320 AAA TGC TGT TCTATT TTC CAG CAA GAG GCT CCC GAG CGA GCA AGC TCA 1065 Lys Cys Cys Ser IlePhe Gln Gln Glu Ala Pro Glu Arg Ala Ser Ser 325 330 335 GTT TAC ACC CGATCC ACT GGG GAG CAG GAA ATA TCT GTG GGC TTG 1110 Val Tyr Thr Arg Ser ThrGly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350 TGACACGGAC TCAAGTGGGCTGGTGACCCA GTCAGAGTTG TGCACATGGC TTAGTTTTCA 1170 TACACAGCCT GGGCTGGGGGTGGGGTGGGA GAGGTCTTTT TTAAAAGGAA GTTACTGTTA 1230 TAGAGGGTCT AAGATTCATCCATTTATTTG GCATCTGTTT AAAGTAGATT AGATCTTTTA 1290 AGCCCATCAA TTATAGAAAGCCAAATCAAA ATATGTTGAT GAAAAATAGC AACCTTTTTA 1350 TCTCCCCTTC ACATGCATCAAGTTATTGAC AAACTCTCCC TTCACTCCGA AAGTTCCTTA 1410 TGTATATTTA AAAGAAAGCCTCAGAGAATT GCTGATTCTT GAGTTTAGTG ATCTGAACAG 1470 AAATACCAAA ATTATTTCAGAAATGTACAA CTTTTTACCT AGTACAAGGC AACATATAGG 1530 TTGTAAATGT GTTTAAAACAGGTCTTTGTC TTGCTATGGG GAGAAAAGAC ATGAATATGA 1590 TTAGTAAAGA AATGACACTTTTCATGTGTG ATTTCCCCTC CAAGGTATGG TTAATAAGTT 1650 TCACTGACTT AGAACCAGGCGAGAGACTTG TGGCCTGGGA GAGCTGGGGA AGCTTCTTAA 1710 ATGAGAAGGA ATTTGAGTTGGATCATCTAT TGCTGGCAAA GACAGAAGCC TCACTGCAAG 1770 CACTGCATGG GCAAGCTTGGCTGTAGAAGG AGACAGAGCT GGTTGGGAAG ACATGGGGAG 1830 GAAGGACAAG GCTAGATCATGAAGAACCTT GACGGCATTG CTCCGTCTAA GTCATGAGCT 1890 GAGCAGGGAG ATCCTGGTTGGTGTTGCAGA AGGTTTACTC TGTGGCCAAA GGAGGGTCAG 1950 GAAGGATGAG CATTTAGGGCAAGGAGACCA CCAACAGCCC TCAGGTCAGG GTGAGGATGG 2010 CCTCTGCTAA GCTCAAGGCGTGAGGATGGG AAGGAGGGAG GTATTCGTAA GGATGGGAAG 2070 GAGGGAGGTA TTCGTGCAGCATATGAGGAT GCAGAGTCAG CAGAACTGGG GTGGATTTGG 2130 TTTGGAAGTG AGGGTCAGAGAGGAGTCAGA GAGAATCCCT AGTCTTCAAG CAGATTGGAG 2190 AAACCCTTGA AAAGACATCAAGCACAGAAG GAGGAGGAGG AGGTTTAGGT CAAGAAGAAG 2250 ATGGATTGGT GTAAAAGGATGGGTCTGGTT TGCAGAGCTT GAACACAGTC TCACCCAGAC 2310 TCCAGGCTGT CTTTCACTGAATGCTTCTGA CTTCATAGAT TTCCTTCCCA TCCCAGCTGA 2370 AATACTGAGG GGTCTCCAGGAGGAGACTAG ATTTATGAAT ACACGAGGTA TGAGGTCTAG 2430 GAACATACTT CAGCTCACACATGAGATCTA GGTGAGGATT GATTACCTAG TAGTCATTTC 2490 ATGGGTTGTT GGGAGGATTCTATGAGGCAA CCACAGGCAG CATTTAGCAC ATACTACACA 2550 TTCAATAAGC ATCAAACTCTTAGTTACTCA TTCAGGGATA GCACTGAGCA AAGCATTGAG 2610 CAAAGGGGTC CCATATAGGTGAGGGAAGCC TGAAAAACTA AGATGCTGCC TGCCCAGTGC 2670 ACACAAGTGT AGGTATCATTTTCTGCATTT AACCGTCAAT AGGCAAAGGG GGGAAGGGAC 2730 ATATTCATTT GGAAATAAGCTGCCTTGAGC CTTAAAACCC ACAAAAGTAC AATTTACCAG 2790 CCTCCGTATT TCAGACTGAATGGGGGTGGG GGGGGCGCCT TAGGTACTTA TTCCAGATGC 2850 CTTCTCCAGA CAAACCAGAAGCAACAGAAA AAATCGTCTC TCCCTCCCTT TGAAATGAAT 2910 ATACCCCTTA GTGTTTGGGTATATTCATTT CAAAGGGAGA GAGAGAGGTT TTTTTCTGTT 2970 CTTTCTCATA TGATTGTGCACATACTTGAG ACTGTTTTGA ATTTGGGGGA TGGCTAAAAC 3030 CATCATAGTA CAGGTAAGGTGAGGGAATAG TAAGTGGTGA GAACTACTCA GGGAATGAAG 3090 GTGTCAGAAT AATAAGAGGTGCTACTGACT TTCTCAGCCT CTGAATATGA ACGGTGAGCA 3150 TTGTGGCTGT CAGCAGGAAGCAACGAAGGG AAATGTCTTT CCTTTTGCTC TTAAGTTGTG 3210 GAGAGTGCAA CAGTAGCATAGGACCCTACC CTCTGGGCCA AGTCAAAGAC ATTCTGACAT 3270 CTTAGTATTT GCATATTCTTATGTATGTGA AAGTTACAAA TTGCTTGAAA GAAAATATGC 3330 ATCTAATAAA AAACACCTTCTAAAATAAAA AAAAAAAAAA AAAAAAAAAA AAA 3383 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 352 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “88C aminoacid sequence” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Asp Tyr GlnVal Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 Ser Glu ProCys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 Leu Pro ProLeu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 Met Leu ValIle Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met 50 55 60 Thr Asp IleTyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 Leu ThrVal Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 Gly AsnThr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110 PheSer Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120 125Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe 130 135140 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser 145150 155 160 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu HisTyr 165 170 175 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe TrpLys Asn 180 185 190 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val LeuPro Leu Leu 195 200 205 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys ThrLeu Leu Arg Cys 210 215 220 Arg Asn Glu Lys Lys Arg His Arg Ala Val ArgLeu Ile Phe Thr Ile 225 230 235 240 Met Ile Val Tyr Phe Leu Phe Trp AlaPro Tyr Asn Ile Val Leu Leu 245 250 255 Leu Asn Thr Phe Gln Glu Phe PheGly Leu Asn Asn Cys Ser Ser Ser 260 265 270 Asn Arg Leu Asp Gln Ala MetGln Val Thr Glu Thr Leu Gly Met Thr 275 280 285 His Cys Cys Ile Asn ProIle Ile Tyr Ala Phe Val Gly Glu Lys Phe 290 295 300 Arg Asn Tyr Leu LeuVal Phe Phe Gln Lys His Ile Ala Lys Arg Phe 305 310 315 320 Cys Lys CysCys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser 325 330 335 Ser ValTyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350 (2)INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1915 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:CDS (B) LOCATION: 362..1426 (ix) FEATURE: (A) NAME/KEY: misc_feature (D)OTHER INFORMATION: /= “88-2B polynucleotide and amino acid sequences”(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATAATAATGA TTATTATATTGTTATCATTA TCTAGCCTGT TTTTTCCTGT TTTGTATTTC 60 TTCCTTTAAA TGCTTTCAGAAATCTGTATC CCCATTCTTC ACCACCACCC CACAACATTT 120 CTGCTTCTTT TCCCATGCCGGGTCATGCTA ACTTTGAAAG CTTCAGCTCT TTCCTTCCTC 180 AATCCTTTTC CTGGCACCTCTGATATGCCT TTTGAAATTC ATGTTAAAGA ATCCCTAGGC 240 TGCTATCACA TGTGGCATCTTTGTTGAGTA CATGAATAAA TCAACTGGTG TGTTTTACGA 300 AGGATGATTA TGCTTCATTGTGGGATTGTA TTTTTCTTCT TCTATCACAG GGAGAAGTGA 360 A ATG ACA ACC TCA CTAGAT ACA GTT GAG ACC TTT GGT ACC ACA TCC 406 Met Thr Thr Ser Leu Asp ThrVal Glu Thr Phe Gly Thr Thr Ser 1 5 10 15 TAC TAT GAT GAC GTG GGC CTGCTC TGT GAA AAA GCT GAT ACC AGA GCA 454 Tyr Tyr Asp Asp Val Gly Leu LeuCys Glu Lys Ala Asp Thr Arg Ala 20 25 30 CTG ATG GCC CAG TTT GTG CCC CCGCTG TAC TCC CTG GTG TTC ACT GTG 502 Leu Met Ala Gln Phe Val Pro Pro LeuTyr Ser Leu Val Phe Thr Val 35 40 45 GGC CTC TTG GGC AAT GTG GTG GTG GTGATG ATC CTC ATA AAA TAC AGG 550 Gly Leu Leu Gly Asn Val Val Val Val MetIle Leu Ile Lys Tyr Arg 50 55 60 AGG CTC CGA ATT ATG ACC AAC ATC TAC CTGCTC AAC CTG GCC ATT TCG 598 Arg Leu Arg Ile Met Thr Asn Ile Tyr Leu LeuAsn Leu Ala Ile Ser 65 70 75 GAC CTG CTC TTC CTC GTC ACC CTT CCA TTC TGGATC CAC TAT GTC AGG 646 Asp Leu Leu Phe Leu Val Thr Leu Pro Phe Trp IleHis Tyr Val Arg 80 85 90 95 GGG CAT AAC TGG GTT TTT GGC CAT GGC ATG TGTAAG CTC CTC TCA GGG 694 Gly His Asn Trp Val Phe Gly His Gly Met Cys LysLeu Leu Ser Gly 100 105 110 TTT TAT CAC ACA GGC TTG TAC AGC GAG ATC TTTTTC ATA ATC CTG CTG 742 Phe Tyr His Thr Gly Leu Tyr Ser Glu Ile Phe PheIle Ile Leu Leu 115 120 125 ACA ATC GAC AGG TAC CTG GCC ATT GTC CAT GCTGTG TTT GCC CTT CGA 790 Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala ValPhe Ala Leu Arg 130 135 140 GCC CGG ACT GTC ACT TTT GGT GTC ATC ACC AGCATC GTC ACC TGG GGC 838 Ala Arg Thr Val Thr Phe Gly Val Ile Thr Ser IleVal Thr Trp Gly 145 150 155 CTG GCA GTG CTA GCA GCT CTT CCT GAA TTT ATCTTC TAT GAG ACT GAA 886 Leu Ala Val Leu Ala Ala Leu Pro Glu Phe Ile PheTyr Glu Thr Glu 160 165 170 175 GAG TTG TTT GAA GAG ACT CTT TGC AGT GCTCTT TAC CCA GAG GAT ACA 934 Glu Leu Phe Glu Glu Thr Leu Cys Ser Ala LeuTyr Pro Glu Asp Thr 180 185 190 GTA TAT AGC TGG AGG CAT TTC CAC ACT CTGAGA ATG ACC ATC TTC TGT 982 Val Tyr Ser Trp Arg His Phe His Thr Leu ArgMet Thr Ile Phe Cys 195 200 205 CTC GTT CTC CCT CTG CTC GTT ATG GCC ATCTGC TAC ACA GGA ATC ATC 1030 Leu Val Leu Pro Leu Leu Val Met Ala Ile CysTyr Thr Gly Ile Ile 210 215 220 AAA ACG CTG CTG AGG TGC CCC AGT AAA AAAAAG TAC AAG GCC ATC CGG 1078 Lys Thr Leu Leu Arg Cys Pro Ser Lys Lys LysTyr Lys Ala Ile Arg 225 230 235 CTC ATT TTT GTC ATC ATG GCG GTG TTT TTCATT TTC TGG ACA CCC TAC 1126 Leu Ile Phe Val Ile Met Ala Val Phe Phe IlePhe Trp Thr Pro Tyr 240 245 250 255 AAT GTG GCT ATC CTT CTC TCT TCC TATCAA TCC ATC TTA TTT GGA AAT 1174 Asn Val Ala Ile Leu Leu Ser Ser Tyr GlnSer Ile Leu Phe Gly Asn 260 265 270 GAC TGT GAG CGG AGC AAG CAT CTG GACCTG GTC ATG CTG GTG ACA GAG 1222 Asp Cys Glu Arg Ser Lys His Leu Asp LeuVal Met Leu Val Thr Glu 275 280 285 GTG ATC GCC TAC TCC CAC TGC TGC ATGAAC CCG GTG ATC TAC GCC TTT 1270 Val Ile Ala Tyr Ser His Cys Cys Met AsnPro Val Ile Tyr Ala Phe 290 295 300 GTT GGA GAG AGG TTC CGG AAG TAC CTGCGC CAC TTC TTC CAC AGG CAC 1318 Val Gly Glu Arg Phe Arg Lys Tyr Leu ArgHis Phe Phe His Arg His 305 310 315 TTG CTC ATG CAC CTG GGC AGA TAC ATCCCA TTC CTT CCT AGT GAG AAG 1366 Leu Leu Met His Leu Gly Arg Tyr Ile ProPhe Leu Pro Ser Glu Lys 320 325 330 335 CTG GAA AGA ACC AGC TCT GTC TCTCCA TCC ACA GCA GAG CCG GAA CTC 1414 Leu Glu Arg Thr Ser Ser Val Ser ProSer Thr Ala Glu Pro Glu Leu 340 345 350 TCT ATT GTG TTT TAGGTCAGATGCAGAAAATT GCCTAAAGAG GAAGGACCAA 1466 Ser Ile Val Phe 355 GGAGATGAAGCAAACACATT AAGCCTTCCA CACTCACCTC TAAAACAGTC CTTCAAACTT 1526 CCAGTGCAACACTGAAGCTC TTGAAGACAC TGAAATATAC ACACAGCAGT AGCAGTAGAT 1586 GCATGTACCCTAAGGTCATT ACCACAGGCC AGGGGCTGGG CAGCGTACTC ATCATCAACC 1646 CTAAAAAGCAGAGCTTTGCT TCTCTCTCTA AAATGAGTTA CCTACATTTT AATGCACCTG 1706 AATGTTAGATAGTTACTATA TGCCGCTACA AAAAGGTAAA ACTTTTTATA TTTTATACAT 1766 TAACTTCAGCCAGCTATTGA TATAAATAAA ACATTTTCAC ACAATACAAT AAGTTAACTA 1826 TTTTATTTTCTAATGTGCCT AGTTCTTTCC CTGCTTAATG AAAAGCTTGT TTTTTCAGTG 1886 TGAATAAATAATCGTAAGCA ACAAAAAAA 1915 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 355 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88-2B amino acid sequence” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Thr Thr Ser Leu Asp Thr Val GluThr Phe Gly Thr Thr Ser Tyr 1 5 10 15 Tyr Asp Asp Val Gly Leu Leu CysGlu Lys Ala Asp Thr Arg Ala Leu 20 25 30 Met Ala Gln Phe Val Pro Pro LeuTyr Ser Leu Val Phe Thr Val Gly 35 40 45 Leu Leu Gly Asn Val Val Val ValMet Ile Leu Ile Lys Tyr Arg Arg 50 55 60 Leu Arg Ile Met Thr Asn Ile TyrLeu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80 Leu Leu Phe Leu Val Thr LeuPro Phe Trp Ile His Tyr Val Arg Gly 85 90 95 His Asn Trp Val Phe Gly HisGly Met Cys Lys Leu Leu Ser Gly Phe 100 105 110 Tyr His Thr Gly Leu TyrSer Glu Ile Phe Phe Ile Ile Leu Leu Thr 115 120 125 Ile Asp Arg Tyr LeuAla Ile Val His Ala Val Phe Ala Leu Arg Ala 130 135 140 Arg Thr Val ThrPhe Gly Val Ile Thr Ser Ile Val Thr Trp Gly Leu 145 150 155 160 Ala ValLeu Ala Ala Leu Pro Glu Phe Ile Phe Tyr Glu Thr Glu Glu 165 170 175 LeuPhe Glu Glu Thr Leu Cys Ser Ala Leu Tyr Pro Glu Asp Thr Val 180 185 190Tyr Ser Trp Arg His Phe His Thr Leu Arg Met Thr Ile Phe Cys Leu 195 200205 Val Leu Pro Leu Leu Val Met Ala Ile Cys Tyr Thr Gly Ile Ile Lys 210215 220 Thr Leu Leu Arg Cys Pro Ser Lys Lys Lys Tyr Lys Ala Ile Arg Leu225 230 235 240 Ile Phe Val Ile Met Ala Val Phe Phe Ile Phe Trp Thr ProTyr Asn 245 250 255 Val Ala Ile Leu Leu Ser Ser Tyr Gln Ser Ile Leu PheGly Asn Asp 260 265 270 Cys Glu Arg Ser Lys His Leu Asp Leu Val Met LeuVal Thr Glu Val 275 280 285 Ile Ala Tyr Ser His Cys Cys Met Asn Pro ValIle Tyr Ala Phe Val 290 295 300 Gly Glu Arg Phe Arg Lys Tyr Leu Arg HisPhe Phe His Arg His Leu 305 310 315 320 Leu Met His Leu Gly Arg Tyr IlePro Phe Leu Pro Ser Glu Lys Leu 325 330 335 Glu Arg Thr Ser Ser Val SerPro Ser Thr Ala Glu Pro Glu Leu Ser 340 345 350 Ile Val Phe 355 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “V28degf2” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 5: GACGGATCCA TYGAYAGRTA CCTGGCYATY GTCC 34 (2)INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “V28degr2” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 6: GCTAAGCTTT TRTAGGGDGT CCAYAAGAGY AA 32 (2)INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88c-r4” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 7: GATAAGCCTC ACAGCCCTGT G 21 (2) INFORMATIONFOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: misc_feature(D) OTHER INFORMATION: /= “88c-rlb” (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 8: GCTAAGCTTG ATGACTATCT TTAATGTC 28 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: misc_feature (D) OTHERINFORMATION: /= “88-2B-3” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:CCCTCTAGAC TAAAACACAA TAGAGAG 27 (2) INFORMATION FOR SEQ ID NO: 10: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /=“88-2B-5” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GCTAAGCTTATCACAGGGAG AAGTGAAATG 30 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “88-2B-f1”(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AGTGCTAGCA GCTCTTCCTG 20 (2)INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88-2B-r1” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 12: CAGCAGCGTT TTGATGATTC 20 (2) INFORMATION FORSEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: misc_feature (D)OTHER INFORMATION: /= “88C-f1” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:TGTGTTTGCT TTAAAAGCC 19 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “88C-r3”(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TAAGCCTCAC AGCCCTG 17 (2)INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “CCCKR1(2)-5 Primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 15: CGTAAGCTTA GAGAAGCCGG GATGGGAA 28(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iv) ANTI-SENSE: YES (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “CCCKR-3Primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GCCTCTAGAG TCAGAGACCAGCAGA 25 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GACAAGCTTCACAGGGTGGA ACAAGATG 28 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GTCTCTAGACCACTTGAGTC CGTGTCA 27 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1059 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1056 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 19: ATG GAC TAT CAA GTG TCA AGT CCA ACC TAT GACATC GAT TAT TAT ACA 48 Met Asp Tyr Gln Val Ser Ser Pro Thr Tyr Asp IleAsp Tyr Tyr Thr 1 5 10 15 TCG GAA CCC TGC CAA AAA ATC AAT GTG AAA CAAATC GCA GCC CGC CTC 96 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln IleAla Ala Arg Leu 20 25 30 CTG CCT CCG CTC TAC TCA CTG GTG TTC ATC TTT GGTTTT GTG GGC AAC 144 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly PheVal Gly Asn 35 40 45 ATA CTG GTC GTC CTC ATC CTG ATA AAC TGC AAA AGG CTGAAA AGC ATG 192 Ile Leu Val Val Leu Ile Leu Ile Asn Cys Lys Arg Leu LysSer Met 50 55 60 ACT GAC ATC TAC CTG CTC AAC CTG GCC ATC TCT GAC CTG CTTTTC CTT 240 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu PheLeu 65 70 75 80 CTT ACT GTC CCC TTC TGG GCT CAC TAT GCT GCT GCC CAG TGGGAC TTT 288 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp AspPhe 85 90 95 GGA AAT ACA ATG TGT CAA CTC TTG ACA GGG CTC TAT TTT ATA GGCTTC 336 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe100 105 110 TTC TCT GGA ATC TTC TTC ATC ATC CTC CTG ACA ATC GAT AGG TACCTG 384 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu115 120 125 GCT ATC GTC CAT GCT GTG TTT GCT TTA AAA GCC AGG ACA GTC ACCTTT 432 Ala Ile Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe130 135 140 GGG GTG GTG ACA AGT GTG ATC ACT TGG GTG GTG GCT GTG TTT GCCTCT 480 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser145 150 155 160 CTC CCA GGA ATC ATC TTT ACC AGA TCT CAG AGA GAA GGT CTTCAT TAC 528 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Arg Glu Gly Leu HisTyr 165 170 175 ACC TGC AGC TCT CAT TTT CCA TAC AGT CAG TAT CAA TTC TGGAAG AAT 576 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp LysAsn 180 185 190 TTT CAG ACA TTA AAG ATG GTC ATC TTG GGG CTG GTC CTG CCGCTG CTT 624 Phe Gln Thr Leu Lys Met Val Ile Leu Gly Leu Val Leu Pro LeuLeu 195 200 205 GTC ATG GTC ATC TGC TAC TCG GGA ATC CTG AAA ACT CTG CTTCGG TGT 672 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu ArgCys 210 215 220 CGA AAC GAG AAG AAG AGG CAC AGG GCT GTG AGG CTT ATC TTCACC ATC 720 Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe ThrIle 225 230 235 240 ATG ATT GTT TAT TTT CTC TTG TGG GCT CCC TAC AAC ATTGTC CTT CTC 768 Met Ile Val Tyr Phe Leu Leu Trp Ala Pro Tyr Asn Ile ValLeu Leu 245 250 255 CTG AAC ACC TTC CAG GAA TTC TTT GGC CTG AAT AAT TGCAGT AGC TCT 816 Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys SerSer Ser 260 265 270 AAC AGG TTG GAC CAA GCC ATG CAG GTG ACA GAG ACT CTTGGG ATG ACA 864 Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu GlyMet Thr 275 280 285 CAC TGC TGC ATC AAC CCC ATC ATC TAT GCC TTT GTC GGGGAG AAG TTC 912 His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gly GluLys Phe 290 295 300 AGA AAC TAC CTC TTA GTC TTC TTC CAA AAG CAC ATT GCCAAA CGC TTC 960 Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile Ala LysArg Phe 305 310 315 320 TGC AAA TGC TGT TCC ATT TTC CAG CAA GAG GCT CCCGAG CGA GCA AGT 1008 Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala Pro GluArg Ala Ser 325 330 335 TCA GTT TAC ACC CGA TCC ACT GGG GAG CAG GAA ATATCT GTG GGC TTG 1056 Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile SerVal Gly Leu 340 345 350 TGA 1059 (2) INFORMATION FOR SEQ ID NO: 20: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 352 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 20: Met Asp Tyr Gln Val Ser Ser Pro Thr Tyr AspIle Asp Tyr Tyr Thr 1 5 10 15 Ser Glu Pro Cys Gln Lys Ile Asn Val LysGln Ile Ala Ala Arg Leu 20 25 30 Leu Pro Pro Leu Tyr Ser Leu Val Phe IlePhe Gly Phe Val Gly Asn 35 40 45 Ile Leu Val Val Leu Ile Leu Ile Asn CysLys Arg Leu Lys Ser Met 50 55 60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala IleSer Asp Leu Leu Phe Leu 65 70 75 80 Leu Thr Val Pro Phe Trp Ala His TyrAla Ala Ala Gln Trp Asp Phe 85 90 95 Gly Asn Thr Met Cys Gln Leu Leu ThrGly Leu Tyr Phe Ile Gly Phe 100 105 110 Phe Ser Gly Ile Phe Phe Ile IleLeu Leu Thr Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Ala Val PheAla Leu Lys Ala Arg Thr Val Thr Phe 130 135 140 Gly Val Val Thr Ser ValIle Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 Leu Pro Gly IleIle Phe Thr Arg Ser Gln Arg Glu Gly Leu His Tyr 165 170 175 Thr Cys SerSer His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn 180 185 190 Phe GlnThr Leu Lys Met Val Ile Leu Gly Leu Val Leu Pro Leu Leu 195 200 205 ValMet Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys 210 215 220Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe Thr Ile 225 230235 240 Met Ile Val Tyr Phe Leu Leu Trp Ala Pro Tyr Asn Ile Val Leu Leu245 250 255 Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys Ser SerSer 260 265 270 Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu GlyMet Thr 275 280 285 His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val GlyGlu Lys Phe 290 295 300 Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His IleAla Lys Arg Phe 305 310 315 320 Cys Lys Cys Cys Ser Ile Phe Gln Gln GluAla Pro Glu Arg Ala Ser 325 330 335 Ser Val Tyr Thr Arg Ser Thr Gly GluGln Glu Ile Ser Val Gly Leu 340 345 350

We claim:
 1. A method of screening for a modulator of humanimmunodeficiency virus (HIV) or simian immunodeficiency virus (SIV)infection, comprising steps of: (a) contacting a first compositioncomprising an 88C receptor polypeptide with a second compositioncomprising a HIV or SIV envelope protein in the presence and absence ofa compound, wherein the 88C receptor polypeptide comprises an amino acidsequence encoded by a nucleotide sequence that hybridizes to thecomplement of nucleotides 55-1110 of SEQ ID NO: 1 or the complement ofnucleotides 1-1056 of SEQ ID NO: 19 under the following stringentconditions: 42° C. in 50% formamide, 5X SSC, 20 mM sodium phosphate, pH6.8 and washing in 0.2X SSC at 55° C.; (b) measuring the interaction ofthe 88C receptor polypeptide with the envelope protein in the presenceand absence of the compound; and (c) screening for a modulator of HIV orSIV infection, wherein reduced or increased interaction of the 88Creceptor polypeptide with the envelope protein in the presence of thecompound versus in the absence of the compound is indicative of thecompound being a modulator of HIV or SIV infection.
 2. A methodaccording to claim 1, wherein the 88C receptor polypeptide comprises theamino acid sequence set forth in SEQ ID NO:
 2. 3. A method according toclaim 2 wherein the envelope protein comprises an HIV envelope protein.4. A method according to claim 1, wherein the 88C receptor polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:
 20. 5. Amethod according to claim 4, wherein the envelope protein comprises anSIV envelope protein.
 6. A method according to claim 1, wherein thefirst composition comprises a cell that has been recombinantly modifiedto express the 88C receptor polypeptide on its surface.
 7. A methodaccording to claim 6, wherein the second composition comprises HIV whichcomprises HIV envelope protein.
 8. A method according to claim 7,wherein the measuring step comprises measuring HIV infection of thecell.
 9. A method according to claim 1, further comprising a step offorming a modulator composition by mixing a modulator identifiedaccording to step (c) with a pharmaceutically acceptable diluent orcarrier.
 10. A method according to claim 9, further comprising a step ofadministering the modulator composition to a mammalian subject, andscreening for modulation of HIV or SIV infection in said subject.
 11. Amethod of screening for a modulator of human immunodeficiency virus(HIV) infection, comprising steps of: (a) contacting a first compositioncomprising an 88-2B receptor polypeptide with a second compositioncomprising a HIV envelope protein, in the presence and absence of acompound, wherein the 88-2B receptor polypeptide comprises an amino acidsequence encoded by a nucleotide sequence that hybridizes to thecomplement of nucleotides 362-1426 of SEQ ID NO:3 under the followingstringent conditions: 42° C. in 50% formamide, 5X SSC, 20 mM sodiumphosphate, pH 6.8 and washing in 0.2X SSC at 55° C.; (b) measuring theinteraction of the 88-2B receptor polypeptide with the HIV envelopeprotein in the presence and absence of the compound; and (c) screeningfor a modulator of HIV infection, wherein reduced or increasedinteraction of the 88-2B receptor polypeptide with the HIV envelopeprotein in the presence of the compound versus in the absence of thecompound is indicative of the compound being a modulator of HIVinfection.
 12. A method according to claim 11, wherein the 88-2Breceptor polypeptide comprises the amino acid sequence set forth in SEQID NO:
 4. 13. A method according to claim 12, wherein the firstcomposition comprises a cell that has been recombinantly modified toexpress the 88-2B receptor polypeptide on its surface.
 14. A methodaccording to claim 13, wherein the second composition comprises a humanimmunodeficiency virus which comprises HIV envelope protein.
 15. Amethod according to claim 14, wherein the measuring step comprisesmeasuring HIV infection of the cell.
 16. A method according to claim 12,further comprising a step of forming a modulator composition by mixing amodulator identified according to step (c) with a pharmaceuticallyacceptable diluent or carrier.
 17. A method according to claim 16,further comprising a step of administering the modulator composition toa mammalian subject, and screening for modulation of HIV infection insaid subject.
 18. A method for detecting human immunodeficiency virus(HIV) infection of cells, comprising steps of: (a) contacting a cellthat has been recombinantly modified to express at least one of humanchemokine receptors 88C and 88-2B with HIV; and (b) detecting HIVinfection of the cell.
 19. A method for inhibiting humanimmunodeficiency virus (HIV) infection of cells, comprising steps of:(a) contacting cells with an antibody to at least one of human chemokinereceptors 88C and 88-2B with HIV; and (b) detecting HIV infection of thecell after said contacting step.